Search

Biomaterial‐associated-infection causes failure of biomaterial implants. Many new biomaterials have been evaluated for their ability to inhibit bacterial colonization and stimulate tissue-cell-integration, but neglect the role of immune cells. This paper compares macrophage phagocytosis of adhering Staphylococcus aureus on cationic-coatings and patterned poly(ethylene)glycol-hydrogels versus common biomaterials and stainless steel in order to identify surface conditions that promote clearance of adhering bacteria. Staphylococci were allowed to adhere and grow on the materials in a parallel-plate-flow-chamber, after which murine macrophages were introduced. From the decrease in the number of adhering staphylococci, phagocytosis-rates were calculated, and total macrophage displacements during an experiment determined. Hydrophilic surfaces had the lowest phagocytosis-rates, while common biomaterials had intermediate phagocytosis-rates. Patterning of poly(ethylene)glycol-hydrogel coatings increased phagocytosis-rates to the level of common biomaterials, while on cationic-coatings phagocytosis-rates remained relatively low. Likely, phagocytosis-rates on cationic coatings are hampered relative to common biomaterials through strong electrostatic binding of negatively-charged macrophages and staphylococci. On polymeric biomaterials and glass, phagocytosis-rates increased with macrophage displacement, while both parameters increased with biomaterial surface hydrophobicity. Thus hydrophobicity is a necessary surface condition for effective phagocytosis. Concluding, next-generation biomaterials should account for surface effects on phagocytosis in order to enhance the ability of these materials to resist biomaterial-associated-infection. [ABSTRACT FROM AUTHOR]

Abstract: We constructed surface-patterned hydrogels using low-energy focused electron beams to locally crosslink poly(ethylene glycol) (PEG) thin films on silanized glass substrates. Derived from electron-beam lithography, this technique was used to create patterned hydrogels with well-defined spatial positions and degrees of swelling. We found that cells of the bacterium Staphylococcus epidermidis adhered to and grew on the silanized glass substrates. These cells did not, however, adhere to surfaces covered by high-swelling lightly crosslinked PEG hydrogels. This finding is consistent with the cell-repulsiveness generally attributed to PEGylated surfaces. In contrast, S. epidermidis cells did adhere to surfaces covered by low-swelling highly crosslinked hydrogels. By creating precise patterns of repulsive hydrogels combined with adhesive hydrogels or with exposed glass substrate, we were able to spatially control the adhesion of S. epidermidis. Significantly, adhesive areas small enough to trap single bacterial cells could be fabricated. The results suggest that the lateral confinement imposed by cell-repulsive hydrogels hindered the cell proliferation and development into larger bacterial colonies. [Copyright &y& Elsevier]

Abstract: Spectroscopic imaging in the scanning transmission electron microscope (STEM) using spatially resolved electron energy-loss spectroscopy (EELS) provides one of the few ways to quantitatively measure the real-space nanoscale morphology of soft materials such as polymers and biological tissue. This paper describes the basic principles of this technique and outlines some of the important attributes that define the achievable spatial resolution. Many soft materials can be differentiated from each other as well as from solvents based on their EELS fingerprints. Applying a multiple least squares (MLS) fitting algorithm using such spectral fingerprints to analyze spatially resolved spectrum datasets enables the quantitative mapping of the different components in a specimen. However, in contrast to TEM studies of many inorganic materials where the spatial resolution is limited principally by the spherical aberration of the objective lens, the spatial resolution associated with the imaging of radiation-sensitive soft materials is limited by the total electron dose to which they can be exposed before suffering irrevocable chemical or structural damage. The Rose criterion provides a simple guide to enhance the so-called dose-limited spatial resolution relevant to soft-materials imaging. By using the low-loss portion of an EELS spectrum where the inelastic scattering cross-sections are highest together with improvements in data-collection efficiency and post-acquisition data processing, the dose-limited resolution in spectrum images of solvated polymers has moved into the sub 10nm regime. This resolution is sufficient to solve important applications-oriented problems associated with hetero interfaces, nanoscale mixing, and nanophase separation. [Copyright &y& Elsevier]

Poly(ethylene glycol), or PEG, is used extensively in biomedical device development, with applications in tissue engineering, ultrahigh-density protein chips, and in vitro microdevices. We highlight some of the many recent advances in PEG hydrogel technology that center on chemical modifications to impart biospecificity and patterning to control structure on micro- and nanoscales characteristic of individual cells and proteins. [Copyright &y& Elsevier]

Imaging nanoscale polymer objects in the Transmission Electron Microscope is difficult, because small polymeric objects interact only weakly with intermediate-energy electrons. Heavy element staining can induce significant amplitude contrast, but stains can introduce artifacts that complicate the structure determination at nanometer length scales. This paper explores transmission electron holography for phase contrast imaging of unstained arborescent graft polystyrene nanoparticles. Holography is able to recover significant phase contrast from these particles despite the fact that there is negligible amplitude contrast. Comparative imaging experiments show that off-axis holography provides substantially higher contrast than that generated by the traditional method of transferring phase information to amplitude information via defocus. This effect is a consequence of different lens contrast-transfer behavior in each of these two imaging approaches. Under kinematical conditions when the appropriate mean inner potential is known, the specimen''s projected thickness can be directly mapped from the holographic phase image to give a measure of the specimen''s three-dimensional shape. Such quantitative imaging shows that individual arborescent graft polystyrene nanoparticles, which are spherical in a good solvent, adopt a flattened shape when deposited on a carbon substrate and allowed to dry. [Copyright &y& Elsevier]

The article presents a study on the phase-contrast transmission electron microscopy imaging of corneodesmosomes (CD) in cryomicrotomed samples of frozen-hydrated porcine skin and the measurement of corneodesmosomal water concentration. It discusses the spatial resolution required for the measurement of corneodesmosomal water concentration, the average water content of CDs in the lower straum corneum.

Abstract Despite extensive engineering of tissue-contacting biomedical devices to control healing, these devices remain susceptible to bacterial colonization, biofilm formation, and chronic infection. The threat of selecting for resistance genes largely precludes sustained antimicrobial elution as a wide-spread clinical solution. In response, self-defensive surfaces have been developed where antimicrobial is released only when and where there is a bacterial challenge. We explore a new self-defensive approach using anionic microgels into which small-molecule cationic antimicrobials are loaded by complexation. We identify conditions where antimicrobial remains sequestered within the microgels for periods as long as weeks. However, bacterial contact triggers release and leads to local bacterial killing. We speculate that the close proximity of bacteria alters the local thermodynamic environment and interferes with the microgel-antimicrobial complexation. The contact-transfer approach does not require bacterial metabolism but instead appears to be driven by differences between the microgels and the bacterial cell envelope where there is a high concentration of negative charge and hydrophobicity. Contact with metabolizing macrophages or osteoblasts is, however, insufficient to trigger antimicrobial release, indicating that contact transfer can be specific to bacteria and suggesting an avenue to biomedical device surfaces that can simultaneously promote healing and resist infection. [ABSTRACT FROM AUTHOR]

A low-dose, high-resolution, electron-diffraction technique has been used to calculate local orientational order parameters from thermotropic liquid crystal polymer (TLCP) fibers. Diffracted intensities are extracted from digital electron diffraction patterns for the orientational order parameter calculation, in a manner similar to that used with X-ray diffraction data. The resolution of local orientation is made possible by electron diffraction as opposed to other methods because of the ability to sample regions as small as 100 nm in diameter. Working within the critical radiative dose for structural damage constrains the ultimate spatial resolution. The signal-to-noise ratio (SNR) of the diffraction data collected at high spatial resolution is low due to the small volume sampled. This work demonstrates the dependence of the orientational order parameter on signal-to-noise effects and the convergence of the incoming electron beam. [ABSTRACT FROM AUTHOR]

The swell ratio is a key parameter characterizing the structure and properties of a hydrogel. In macroscopic gels, the swell ratio can be determined by straightforward measurements of the gel weight in the dry and hydrated states. However, measuring the swell ratio characteristic of microgels (gel particles with dimensions of 0.1–100 μm) is substantially more challenging because of their small size and polydispersity. We use cryo-scanning electron microscopy (cryo-SEM) to measure microgel diameter both in the frozen-hydrated and fully dry states using pH-responsive poly(ethylene glycol)-co-acrylic acid microgels. The volume swell ratios characteristic of the various microgels are essentially the same as those measured from otherwise-identical macroscopic gels. Hence we can conclude that, at least in this case where the macroscopic gel and microgel synthesis methods are similar, the simple measurement of a macroscopic swell ratio provides a reasonable approximation to the microgel swell ratio. [ABSTRACT FROM AUTHOR]

We report on negatively charged layer-by-layer (LbL) hydrogel films, which turn hydrophobic and bactericidal in response to bacteria-induced acidification of the medium. Single-component hydrogel thin films, abbreviated as PaAA LbL s, consisting of chemically crosslinked poly(2-alkylacrylic acids) (PaAAs) with varying hydrophobicity [polymethacrylic acid (PMAA), poly(2-ethylacrylic acid) (PEAA), poly(2-n-propylacrylic acid) (PPAA) or poly(2-n-butylacrylic acid) (PBAA)]. With increasing polyacid hydrophobicity, the hydrogel films showed a decrease in water uptake and an increase in elastic modulus. Both parameters were strongly dependent on pH. At pH 7.4, hydrogels of higher hydrophobicity were more resistant to colonization by Staphylococcus epidermidis , with the PBAA coating showing almost negligible colonization. As the medium became more acidic due to bacterial proliferation, the more hydrophobic PEAA LbL , PPAA LbL and PBAA LbL hydrogels became dehydrated and killed bacteria upon contact with the surface. The killing efficiency was strongly enhanced by the polymer hydrophobicity. The films remained cytocompatible with human osteoblasts, as indicated by the MTS assay and live/dead staining. Our approach exploits bacteria-responsive properties of the coating itself without the involvement of potentially toxic cationic polymers or the release of antimicrobial agents. These coatings thus demonstrate a novel approach to the antibacterial protection of tissue-contacting biomedical-device surfaces. [ABSTRACT FROM AUTHOR]

Abstract: Surfaces with cell adhesiveness modulated at micro length scales can exploit differences between tissue/bacterial cell size, membrane/wall plasticity, and adhesion mechanisms to differentially control tissue-cell/material and bacteria/material interactions. This study explores the short-term interactions of Staphylococcus aureus and osteoblast-like cells with surfaces consisting of cell-adhesive circular patches (1–5 μm diameter) separated by non-adhesive electron-beam patterned poly(ethylene glycol) hydrogel thin films at inter-patch distances of 0.5–10 μm. Osteoblast-like U2OS cells both bind to and spread on the modulated surfaces, in some cases when the cell-adhesive area comprises only 9% of the total surface and in several cases at least as well as on the continuously adhesive control surfaces. In contrast, S. aureus adhesion rates are 7–20 times less on the modulated surfaces than on the control surfaces. Furthermore, the proliferation of those bacteria that do adhere is inhibited by the lateral confinement imposed by the non-adhesive boundaries surrounding each patch. These findings suggest a new approach to create biomaterial surfaces that may promote healing while simultaneously reducing the probability of infection. [Copyright &y& Elsevier]

Abstract: The surface roughness of metallic orthopaedic implants has typically been used to influence osseointegration and spatially control load transfer to the surrounding bone. Because of the increasing recognition of biomaterials-associated infection as a leading implant failure mode, we are interested to know the relative importance of roughness not only on surface–osteoblast interactions but also on surface–bacteria interactions. This in vitro study thus compares the effects of surface topography on Staphylococcus epidermidis and human osteoblast behavior using four clinically relevant titanium surface finishes: polished, satin, grit-blasted and plasma-sprayed. Important differences between these surfaces are manifested not only by their vertical roughness parameters but also by the lateral length scales over which topographic fluctuations occur. We find that S. epidermidis adhesion and growth is substantially higher on the satin and grit-blasted surfaces than on the polished or plasma-sprayed surfaces. The former are both substantially rougher at length scales comparable to that of bacteria. In contrast, based on imaging and biochemical assays of proliferation, differentiation and matrix formation, we find that desirable osteoblast–surface interactions are maximized on plasma-sprayed surfaces and minimized on satin-finished surfaces. We attribute these differences to the fact that the plasma-sprayed surface is relatively smooth compared to the size of an individual osteoblast, while the satin surface is rough at this length scale. These findings indicate that both the vertical and lateral character of surface roughness can be modified to not only optimize implant–bone interactions but to simultaneously minimize implant–bacteria interactions. [ABSTRACT FROM AUTHOR]

Abstract: We engineered surfaces that permit the adhesion and directed growth of neuronal cell processes but that prevent the adhesion of astrocytes. This effect was achieved based on the spatial distribution of sub-micron-sized cell-repulsive poly(ethylene glycol) [PEG] hydrogels patterned on an otherwise cell-adhesive substrate. Patterns were identified that promoted cellular responses ranging from complete non-attachment, selective attachment, and directed growth at both cellular and subcellular length scales. At the highest patterning density where the individual hydrogels almost overlapped, there was no cellular adhesion. As the spacing between individual hydrogels was increased, patterns were identified where neurites could grow on the adhesive surface between hydrogels while astrocytes were unable to adhere. Patterns such as lines or arrays were identified that could direct the growth of these subcellular neuronal processes. At higher hydrogel spacings, both neurons and astrocytes adhered and grew in a manner approaching that of unpatterned control surfaces. Patterned lines could once again direct growth at cellular length scales. Significantly, we have demonstrated that the patterning of sub-micron/nano scale cell-repulsive features at microscale lengths on an otherwise cell-adhesive surface can differently control the adhesion and growth of cells and cell processes based on the difference in their characteristic sizes. This concept could potentially be applied to an implantable nerve-guidance device that would selectively enable regrowing axons to bridge a spinal-cord injury without interference from the glial scar. [Copyright &y& Elsevier]

Abstract: Tailoring the surfaces of a nanocontainer with polymer brushes that have different affinities to the components of a phase-separating polymer blend should impart self-directing properties to the nanocontainers. Such nanocontainers could then be used to deliver a variety of functional species in tunable amounts and in a site-specific manner to polymer systems. This paper describes the surface modification, subsequent characterization of nanocontainers derived from ferritin, and the effects of surface modification on their self-directing properties in a binary phase-separating homopolymer blend. Wild ferritin was either PEGylated or alkylated by zero-length cross-linking to its surface carboxylate groups that were activated by carbodiimide. Modification was confirmed by ion-exchange chromatography, ζ-potential measurement, and electrospray ionization mass spectrometry. FT-IR spectrometry was used to quantify the extent of PEGylation by ratioing the intensity of the C–O–C asymmetric stretching vibration from the grafted PEG to that of the carbonyl stretching vibration (amide I band) from the protein. Importantly, modified ferritin was soluble in the organic solvent dichloromethane (DCM). Modified ferritin was introduced into a polymer blend of hydrophobic and hydrophilic polymers made up of poly(desaminotyrosyl tyrosine dodecyl ester carbonate) (PDTD) and PEG by solvent casting from solution in the common solvent DCM. Polymer thin films with an average thickness of ∼200μm were obtained upon evaporation of the solvent. Transmission electron micrographs of microtomed polymer films demonstrated remarkable selectivity of PEGylated ferritin to PEG domains, while alkylated ferritin self-directs to the PDTD matrix. [Copyright &y& Elsevier]