Anti-biofouling properties of poly(dimethyl siloxane) with RAFT photopolymerized acrylate/methacrylate surface grafts against model marine organisms.

Biofouling of man-made surfaces by marine organisms is a global problem with both financial and environmental consequences. However, the development of non-toxic anti-biofouling coatings is challenged by the diversity of fouling organisms. One possible solution leverages coatings composed of diverse chemical constituents. Reversible addition-fragmentation chain-transfer (RAFT) photopolymerization was used to modify poly(dimethylsiloxane) (PDMSe) surfaces with polymeric grafts composed of three successive combinations of acrylamide, acrylic acid, and hydroxyethyl methacrylate. RAFT limited conflicting variables and allowed for the effect of graft chemistry to be isolated. While all compositions enhanced the anti-biofouling performance compared with the PDMSe control, the ternary, amphiphilic copolymer was the most effective with 98% inhibition of the attachment of zoospores of the green alga Ulva linza, 94% removal of cells of the diatom Navicula incerta, and 62% removal of cells of the bacterium Cellulophaga lytica. However, none of the graft compositions tested were able to mitigate reattachment of adult barnacles, Amphibalanus amphitrite.

Engineered Chemical Nanotopographies: Reversible Addition-Fragmentation Chain-Transfer Mediated Grafting of Anisotropic Poly(acrylamide) Patterns on Poly(dimethylsiloxane) To Modulate Marine Biofouling.

Effectively negating the deleterious impact of marine biofouling on the world's maritime fleet in an environmentally conscientious manner presents a difficult challenge due to a variety of factors including the complexity and diversity of fouling species and the differing surface adhesion strategies. Understanding how surface properties relate to biofouling can inform and guide the development of new antibiofouling coatings to address this challenge. Herein, we report on the development of a living photopolymerization strategy used to tailor the surface properties of silicone rubber using controlled anisotropic poly(acrylamide) patterns and the resulting antibiofouling efficacy of these surfaces against zoospores of the model marine fouling organism, Ulva linza. Chemical patterns were fabricated using reversible addition-fragmentation chain-transfer (RAFT) living polymerization in conjunction with photolithography. Pattern geometries were inspired by the physical (i.e., nonchemical) Sharklet engineered microtopography system that has been shown to be effective against the same model organism. Sharklet chemical patterns and analogous parallel channels were fabricated in sizes ranging from 2 to 10 µm in the lateral dimension with tailorable feature heights ranging from tens to hundreds of nanometers. Nonpatterned, chemically grafted poly(acrylamide) silicone surfaces inhibited algal spore attachment density by 59% compared to the silicone control; however, attachment density on chemical nanotopographies was not statistically different from the control. While these results indicate that the chemical nanotopographies chosen do not represent an effective antibiofouling coating, it was found that the Sharklet pattern geometry, when sized below the 5 µm critical attachment size of the spores, significantly reduced the algal spore density compared to the equally sized channel geometry. These results indicate that specific chemical geometry of the proper sizing can impact the behavior of the algal spores and could be used to further study the mechanistic behavior of biofouling organisms.

Effects of inter-organism interactions in biofouling on microtopographic surfaces.

An extended model of the surface energetic attachment (SEA) model is introduced to study the fouling of marine organisms on microtopographic surfaces, taking into account the excluded volume interaction and the attraction between the organisms. It is shown that the excluded volume interaction leads to changes in the site-typed attachment probabilities which increase with the average spore density on the surface. As a result of these changes, the spore density map is flattened under very high density fouling. The attractive interaction on the other hand leads to aggregation of spores and the average aggregate size increased with the strength of attraction. The model can be mapped to a specific experiment to determine the attachment energy parameters. In contrast to various prior empirical approaches, the extended SEA model is rigorous from the statistical mechanics viewpoint, thus it provides a reliable tool for studying complex attachment behaviors of microorganisms on topographic surfaces.

Marine anti-biofouling efficacy of amphiphilic poly(coacrylate) grafted PDMSe: effect of graft molecular weight.

There is currently strong motivation due to ecological concerns to develop effective anti-biofouling coatings that are environmentally benign, durable, and stable for use by the maritime industry. The antifouling (AF) and fouling-release (FR) efficacy of amphiphilic, charged copolymers composed of ~52% acrylamide, ~34% acrylic acid, and ~14% methyl acrylate grafted to poly(dimethyl siloxane) (PDMSe) surfaces were tested against zoospores of the green alga Ulva linza and the diatom Navicula incerta. The biofouling response to molecular weight variation was analyzed for grafts ranging from ~100 to 1,400 kg mol-1, The amphiphilic coatings showed a marked improvement in the FR response, with a 55% increase in the percentage removal of diatoms and increased AF efficacy, with 92% reduction in initial attachment density of zoospores, compared to PDMSe controls. However, graft molecular weight, in the range tested, was statistically insignificant. Grafting copolymers to PDMSe embossed with the Sharklet™ microtopography did not produce enhanced AF efficacy.

Engineered antifouling microtopographies: surface pattern effects on cell distribution.

Microtopography has been observed to lead to altered attachment behavior for marine fouling organisms; however, quantification of this phenomenon is lacking in the scientific literature. Here, we present quantitative measurement of the disruption of normal attachment behavior of the fouling algae Ulva linza by antifouling microtopographies. The distribution of the diatom Navicula incerta was shown to be unaffected by the presence of topography. The radial distribution function was calculated for both individual zoospores and cells as well as aggregates of zoospores from attachment data for a variety topographic configurations and at a number of different attachment densities. Additionally, the screening distance and maximum values were mapped according to the location of zoospore aggregates within a single unit cell. We found that engineered topographies decreased the distance between spore aggregates compared to that for a smooth control surface; however, the distributions for individual spores were unchanged. We also found that the local attachment site geometry affected the screening distance for aggregates of zoospores, with certain geometries decreasing screening distance and others having no measurable effect. The distribution mapping techniques developed and explored in this article have yielded important insight into the design parameters for antifouling microtopographies that can be implemented in the next generation of antifouling surfaces.

Engineered antifouling microtopographies: an energetic model that predicts cell attachment.

We have developed a model for the prediction of cell attachment to engineered microtopographies based on two previous models: the attachment point theory and the engineered roughness index (ERI) model. The new surface energetic attachment (SEA) model is based on both the properties of the cell-material interface and the size and configuration of the topography relative to the organism. We have used Monte Carlo simulation to examine the SEA model's ability to predict relative attachment of the green alga Ulva linza to different locations within a unit cell. We have also compared the predicted relative attachment for Ulva linza, the diatom Navicula incerta, the marine bacterium Cobetia marina, and the barnacle cyprid Balanus amphitrite to a wide variety of microtopographies. We demonstrate good correlation between the experimental results and the model results for all tested experimental data and thus show the SEA model may be used as a powerful indicator of the efficacy for antifouling topographies.

Surface modification of silicate glass using 3-(mercaptopropyl)trimethoxysilane for thiol-ene polymerization.

A thiol-ene polymerization was accomplished on silicate glass slides to graft a series of homopolymers and copolymers using 3-(mercaptopropyl)trimethoxysilane (MTS) as both a silane coupling agent and initiator. MTS was initially covalently bonded to an acid cleaned glass surface via a classical sol-gel reaction. Poly(acrylic acid) (PAA), poly(acrylamide) (PAAm), poly(methyl acrylate) (PMA), poly(acrylamido-2-methyl-propanesulfonic acid) (PAMPS), and the copolymer poly(AA-co-AAm-co-MA-co-AMPS) were grafted from the thiol group of MTS. The surface chemistry of the MTS modified slides and polymer grafts was characterized with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Surface texture was evaluated with tapping mode atomic force microscopy (TM-AFM). The Owens-Wendt-Kaelble (OWK) and Lifshitz-van der Waals acid-base (LW-AB) methods were used to evaluate surface energies by sessile drop contact angle method. The synthetic approach demonstrated a facile, rapid method for grafting to glass surfaces.

Antifouling performance of cross-linked hydrogels: refinement of an attachment model.

Poly(ethylene glycol) dimethacrylate (PEGDMA), PEGDMA-co-glycidyl methacrylate (PEGDMA-co-GMA), and PEGDMA-co-hydroxyethyl methacrylate (PEGDMA-co-HEMA) hydrogels were polymerized using ammonium persulfate and ascorbic acid as radical initiators. Surface energies of the hydrogels and a standard, poly(dimethylsiloxane) elastomer (PDMSe), were characterized using captive bubble and sessile drop measurements, respectively (γ = 52 mN/m, γ(0) = 19 mN/m). The chemical composition of the hydrogels was characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. All three hydrogel compositions reduced significantly (p = 0.05) initial attachment of zoospores of the green alga Ulva linza (up to 97%), cells of the diatom Navicula incerta (up to 58%) and the bacterium Cobetia marina (up to 62%), compared to a smooth PDMSe standard. A shear stress (45 Pa), generated in a water channel, eliminated up to 95% of the initially attached cells of Navicula from the smooth hydrogel surfaces relative to smooth PDMSe surfaces. Compared to the PDMSe standard, 79% of the cells of C. marina were removed from all smooth hydrogel compositions when exposed to a 50 Pa wall shear stress. Attachment of spores of the green alga Ulva to microtopographies replicated in PEGDMA-co-HEMA was also evaluated. The Sharklet AF microtopography patterned, PEGDMA-co-HEMA surfaces reduced attachment of spores of Ulva by 97% compared to a smooth PDMSe standard. The attachment densities of spores to engineered microtopographies in PDMSe and PEGDMA-co-HEMA were shown to correlate with a modified attachment model through the inclusion of a surface energy term. Attachment densities of spores of Ulva to engineered topographies replicated in a material other than PDMSe are now correlated with the attachment model (R(2) = 0.80).

Engineered antifouling microtopographies: kinetic analysis of the attachment of zoospores of the green alga Ulva to silicone elastomers.

Microtopography has been demonstrated as an effective deterrent to biofouling. The majority of published studies are fixed-time assays that raise questions regarding the kinetics of the attachment process. This study investigated the time-dependent attachment density of zoospores of Ulva, in a laboratory assay, on a micropatterned and smooth silicone elastomer. The attachment density of zoospores was reduced on average 70-80% by the microtopography relative to smooth surfaces over a 4 h exposure. Mapping the zoospore locations on the topography revealed that they settled preferentially in specific, recessed areas of the pattern. The kinetic data fit, with high correlation (r(2) > 0.9), models commonly used to describe the adhesion of bacteria to surfaces. The grouping of spores on the microtopography indicated that the pattern inhibited the ability of attached spores to recruit neighbors. This study demonstrates that the antifouling mechanism of topographies may involve disruption of the cooperative effects exhibited by fouling organisms such as Ulva.

Microtopographical patterns promote different responses in fibroblasts and Schwann cells: A possible feature for neural implants.

The chronic reliability of bioelectronic neural interfaces has been challenged by foreign body reactions (FBRs) resulting in fibrotic encapsulation and poor integration with neural tissue. Engineered microtopographies could alleviate these challenges by manipulating cellular responses to the implanted device. Parallel microchannels have been shown to modulate neuronal cell alignment and axonal growth, and Sharklet™ microtopographies of targeted feature sizes can modulate bio-adhesion of an array of bacteria, marine organisms, and epithelial cells due to their unique geometry. We hypothesized that a Sharklet™ micropattern could be identified that inhibited fibroblasts partially responsible for FBR while promoting Schwann cell proliferation and alignment. in vitro cell assays were used to screen the effect of Sharklet™ and channel micropatterns of varying dimensions from 2 to 20 µm on fibroblast and Schwann cell metrics (e.g., morphology/alignment, nuclei count, metabolic activity), and a hierarchical analysis of variance was used to compare treatments. In general, Schwann cells were found to be more metabolically active and aligned than fibroblasts when compared between the same pattern. 20 µm wide channels spaced 2 µm apart were found to promote Schwann cell attachment and alignment while simultaneously inhibiting fibroblasts and warrant further in vivo study on neural interface devices. No statistically significant trends between cellular responses and geometrical parameters were identified because mammalian cells can change their morphology dependent on their environment in a manner dissimilar to bacteria. Our results showed although surface patterning is a strong physical tool for modulating cell behavior, responses to micropatterns are highly dependent on the cell type.

Engineered antifouling microtopographies: the role of Reynolds number in a model that predicts attachment of zoospores of Ulva and cells of Cobetia marina.

A correlation between the attachment density of cells from two phylogenetic groups (prokaryotic Bacteria and eukaryotic Plantae), with surface roughness is reported for the first time. The results represent a paradigm shift in the understanding of cell attachment, which is a critical step in the biofouling process. The model predicts that the attachment densities of zoospores of the green alga, Ulva, and cells of the marine bacterium, Cobetia marina, scale inversely with surface roughness. The size and motility of the bacterial cells and algal spores were incorporated into the attachment model by multiplying the engineered roughness index (ERI(II)), which is a representation of surface energy, by the Reynolds number (Re) of the cells. The results showed a negative linear correlation of normalized, transformed attachment density for both organisms with ERI(II) x Re (R(2) = 0.77). These studies demonstrate for the first time that organisms respond in a uniform manner to a model, which incorporates surface energy and the Reynolds number of the organism.

Engineered antifouling microtopographies: mapping preferential and inhibitory microenvironments for zoospore attachment.

An algorithm was developed and implemented to map the locations of attached spores of Ulva linza on patterned surfaces. Using this mapping algorithm, spore preference among regions within a pattern can be detected and quantified. Settlement maps of spores on patterned topographies from several assays showed clear preferences in spore settlement. Over 94% of the spores attached within the depressed regions on the surfaces, including a surface containing pits instead of protruding features. The spores attached primarily at the intersections of several features, with over half and up to 96% of spores settling in these regions. The highest spore densities occurred at intersections where the features were most dissimilar. In contrast, the location of attached beads on the surfaces was nearly uniform across the surface. Identification of preferential attachment locations allows for the study of localized properties that influence cell behavior and aids in the development of new surfaces to control cell-surface interactions.

A model that predicts the attachment behavior of Ulva linza zoospores on surface topography.

A predictive model for the attachment of spores of the green alga Ulva on patterned topographical surfaces was developed using a constant refinement approach. This 'attachment model' incorporated two historical data sets and a modified version of the previously-described Engineered Roughness Index. Two sets of newly-designed surfaces were used to evaluate the effect of two components of the model on spore settlement. Spores attached in fewer numbers when the area fraction of feature tops increased or when the number of distinct features in the design increased, as predicted by the model. The model correctly predicted the spore attachment density on three previously-untested surfaces relative to a smooth surface. The two historical data sets and two new data sets showed high correlation (R(2) = 0.88) with the model. This model may be useful for designing new antifouling topographies.

Surface texture limits transfer of S. aureus, T4 bacteriophage, influenza B virus and human coronavirus.

Spread of pathogens on contaminated surfaces plays a key role in disease transmission. Surface technologies that control pathogen transfer can help control fomite transmission and are of great interest to public health. Here, we report a novel bead transfer method for evaluating fomite transmission in common laboratory settings. We show that this method meets several important criteria for quantitative test methods, including reasonableness, relevancy, resemblance, responsiveness, and repeatability, and therefore may be adaptable for standardization. In addition, this method can be applied to a wide variety of pathogens including bacteria, phage, and human viruses. Using the bead transfer method, we demonstrate that an engineered micropattern limits transfer of Staphylococcus aureus by 97.8% and T4 bacteriophage by 93.0% on silicone surfaces. Furthermore, the micropattern significantly reduces transfer of influenza B virus and human coronavirus on silicone and polypropylene surfaces. Our results highlight the potential of using surface texture as a valuable new strategy in combating infectious diseases.

Potential for tunable static and dynamic contact angle anisotropy on gradient microscale patterned topographies.

Translationally symmetric topographies can be designed to induce anisotropy of static and dynamic contact angles. The validity of ignoring directionality of topography in contact angle characterization was evaluated using microscale patterned topographies. Seven patterned topographies comprising elongated discontinuous microfeatures oriented along parallel paths and one topography comprising ridges were fabricated in a poly(dimethyl siloxane) elastomer (PDMSe). The static contact angle, advancing contact angle, receding contact angle, contact angle hysteresis, and slip angle were measured using water on each surface at three in-plane perspectives, with respect to the feature orientation. Static and dynamic contact angle anisotropies were investigated on the topographies to evaluate the effect of discontinuities along the feature lengths on the anisotropy that has been shown on channels or ridges in previous reports. Discontinuous feature topographies exhibited a statistically significant anisotropy of 2 degrees-6 degrees between the perpendicular and parallel directions, with respect to the static and dynamic contact angles. The ridges topography exhibited much larger 5 degrees-42 degrees anisotropy in the contact angles. The discontinuities along the feature lengths greatly reduced, but did not eliminate, the anisotropies compared to the ridges. This evidence of contact angle anisotropy indicates a need to identify the orientation of topography, in relation to contact angle measurements. It also implies a need to consider directionality in the design of microfluidic devices and self-cleaning surfaces.

Surface micropattern reduces colonization and medical device-associated infections.

PURPOSE: Surface microtopography offers a promising approach for infection control. The goal of this study was to provide evidence that micropatterned surfaces significantly reduce the potential risk of medical device-associated infections. METHODOLOGY: Micropatterned and smooth surfaces were challenged in vitro against the colonization and transference of two representative bacterial pathogens - Staphylococcus aureus and Pseudomonas aeruginosa. A percutaneous rat model was used to assess the effectiveness of the micropattern against device-associated S. aureus infections. After the percutaneous insertion of silicone rods into (healthy or immunocompromised) rats, their backs were inoculated with S. aureus. The bacterial burdens were determined in tissues under the rods and in the spleens. RESULTS: The micropatterns reduced adherence by S. aureus (92.3 and 90.5 % reduction for flat and cylindrical surfaces, respectively), while P. aeruginosa colonization was limited by 99.9 % (flat) and 95.5 % (cylindrical). The micropatterned surfaces restricted transference by 95.1 % for S. aureus and 94.9 % for P. aeruginosa, compared to smooth surfaces. Rats with micropatterned devices had substantially fewer S. aureus in subcutaneous tissues (91 %) and spleens (88 %) compared to those with smooth ones. In a follow-up study, immunocompromised rats with micropatterned devices had significantly lower bacterial burdens on devices (99.5 and 99.9 % reduction on external and internal segments, respectively), as well as in subcutaneous tissues (97.8 %) and spleens (90.7 %) compared to those with smooth devices. CONCLUSION: Micropatterned surfaces exhibited significantly reduced colonization and transference in vitro, as well as lower bacterial burdens in animal models. These results indicate that introducing this micropattern onto surfaces has high potential to reduce medical device-associated infections.

Evaluation of a bilayered, micropatterned hydrogel dressing for full-thickness wound healing.

Nearly 12 million wounds are treated in emergency departments throughout the United States every year. The limitations of current treatments for complex, full-thickness wounds are the driving force for the development of new wound treatment devices that result in faster healing of both dermal and epidermal tissue. Here, a bilayered, biodegradable hydrogel dressing that uses microarchitecture to guide two key steps in the proliferative phase of wound healing, re-epithelialization, and revascularization, was evaluated in vitro in a cell migration assay and in vivo in a bipedicle ischemic rat wound model. Results indicate that the Sharklet™-micropatterned apical layer of the dressing increased artificial wound coverage by up to 64%, P = 0.024 in vitro. In vivo evaluation demonstrated that the bilayered dressing construction enhanced overall healing outcomes significantly compared to untreated wounds and that these outcomes were not significantly different from a leading clinically available wound dressing. Collectively, these results demonstrate high potential for this new dressing to effectively accelerate wound healing.

Micropatterned Endotracheal Tubes Reduce Secretion-Related Lumen Occlusion.

Tracheal intubation disrupts physiological homeostasis of secretion production and clearance, resulting in secretion accumulation within endotracheal tubes (ETTs). Novel in vitro and in vivo models were developed to specifically recapitulate the clinical manifestations of ETT occlusion. The novel Sharklet™ micropatterned ETT was evaluated, using these models, for the ability to reduce the accumulation of both bacterial biofilm and airway mucus compared to a standard care ETT. Novel ETTs with micropattern on the inner and outer surfaces were placed adjacent to standard care ETTs in in vitro biofilm and airway patency (AP) models. The primary outcome for the biofilm model was to compare commercially-available ETTs (standard care and silver-coated) to micropatterned for quantity of biofilm accumulation. The AP model's primary outcome was to evaluate accumulation of artificial airway mucus. A 24-h ovine mechanical ventilation model evaluated the primary outcome of relative quantity of airway secretion accumulation in the ETTs tested. The secondary outcome was measuring the effect of secretion accumulation in the ETTs on airway resistance. Micropatterned ETTs significantly reduced biofilm by 71% (p = 0.016) compared to smooth ETTs. Moreover, micropatterned ETTs reduced lumen occlusion, in the AP model, as measured by cross-sectional area, in distal (85%, p = 0.005), middle (84%, p = 0.001) and proximal (81%, p = 0.002) sections compared to standard care ETTs. Micropatterned ETTs reduced the volume of secretion accumulation in a sheep model of occlusion by 61% (p < 0.001) after 24 h of mechanical ventilation. Importantly, micropatterned ETTs reduced the rise in ventilation peak inspiratory pressures over time by as much as 49% (p = 0.005) compared to standard care ETTs. Micropatterned ETTs, demonstrated here to reduce bacterial contamination and mucus occlusion, will have the capacity to limit complications occurring during mechanical ventilation and ultimately improve patient care.

Biocomposites with tunable properties from poly(lactic acid)-based copolymers and carboxymethyl cellulose via ionic assembly.

Biocomposites with tunable properties were successfully prepared through ionic assembly between anionic carboxymethyl cellulose (CMC) and cationic copolymers (quaternized poly(l-lactide)-block-poly N,N-dimethylamino-2-ethyl methacrylate) (PLA-b-PDMAEMA). The quaternized PDMAEMA segment not only works as a compatibilizer between hydrophilic CMC and hydrophobic PLA, but also acts as a lubricant between these two rigid biopolymers. The (1)H NMR (nuclear magnetic resonance) spectra demonstrated successful synthesis of PLA-b-PDMAEMA with controlled molecular weight of PDMAEMA segment. The results from scanning electronic microscopy (SEM) and Fourier transform infrared spectrometry (FTIR) verified the interaction between quaternized copolymer micelles and anionic CMC networks. The resultant biocomposite could form a transparent and uniform film after casting. Both storage moduli and maximum degradation temperature of PLA/CMC composites were increased with the reduction of molecular weight of PDMAEMA segments. It suggests that the properties of biocomposite materials can be tailored by adjusting the chain length of inclusive PDMAEMA segment.

Gecko Adhesion on Wet and Dry Patterned Substrates.

Perhaps one of the most astounding characteristics of the gecko adhesive system is its versatility. Geckos can locomote across complex substrates in a variety of conditions with apparent ease. In contrast, many of our synthetic pressure sensitive adhesives fail on substrates that are dirty, wet or rough. Although many studies have investigated the effect of environmental challenges on performance, the interaction of multiple, potentially compromising variables is studied less often. Here we focus on substrate structure and surface water, both of which are highly relevant to the biological system and to synthetic design. To do this we utilized a highly controlled, patterned substrate (Sharklet®, by Sharklet® Technologies Inc.). This allowed us to test independently and jointly the effects of reduced surface area substrates, with a defined pattern, on adhesion in both air and water. Our results show that adhesion is not significantly impaired in air, whereas surface area and pattern significantly affect adhesion in water. These findings highlight the need to study multiple parameters that are relevant to the gecko adhesive system to further improve our understanding of the biological system and to design better, more versatile synthetics.