Nanoscale Surface Topography Research Articles

Understanding the role of polymer surface nanoscale topography on inhibiting bacteria adhesion and growth

Event Abstract Back to Event Understanding the role of polymer surface nanoscale topography on inhibiting bacteria adhesion and growth Luting Liu1 and Thomas J. Webster1, 2 1 Northeastern University, Department of Chemical Engineering, United States 2 King Abdulaziz University, Center of Excellence for Advanced Materials Research, Saudi Arabia Introduction: Currently, the use of nanostructured materials, especially materials with nanofeatured topographies, which have more surface area, altered surface energy, enhanced select protein adsorption, selectively increased desirable cell functions while simultaneously decreasing competitive cell functions, seem to be among the most promising ways for reducing initial bacteria attachment, biofilm formation and infection[1]. Also, natural surfaces, such as cicada wing surfaces, appear to be bactericidal to Pseudomonas aeruginosa, which was thought to be due to the nanostructure surface of the wing rather than a chemical effect[2]. Motivated by these findings, this study aimed to modify the raw surface of a catheter composed of polydimethylsiloxane (PDMS) to possess antibacterial nanostructures, and then to develop a mathematical model that can correlate nanosurface roughness with protein adsorption and bacteria adhesion. Materials and Methods: Here, we present a simple method to prepare a nano-patterned PDMS replica by using nanotubular anodized titanium as the template (Fig. 1)[3]. The surface morphogy and elemental composition of PDMS were characterized by SEM, AFM, and XPS. In vitro bacterial studies using Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were conducted to assess the effectiveness of the nano-patterned PDMS (nano-PDMS) at inhibiting bacteria adhesion and growth. In addition, human fibroblast (ATCC, CCL-110) and endothelial cell (Life Technologies) MTT assays were conducted to determine the toxicity of the nanostructure to mammalian cells. To study the mechanism of these nanostructured features towards inhibiting bacteria adhesion and growth, the adsorption of proteins from tryptic soy broth that influence bacteria functions were determined. Results and Discussion: As expected, the nano-patterned structures were fabricated successfully on the surface of PDMS without changing its surface chemistry. In vitro studies indicated that nano-PDMS inhibited the adhesion and growth of both two bacteria after 48h compared with plain-PDMS. Moreover, data suggested the effectiveness of bacteria inhibition reached above 50%, all without employing antibiotics (Fig. 2). It was also found that nano-PDMS increased both fibroblast and endothelial cell adhesion after 4h of treatment. A bicinchoninic acid protein assay kit was used to quantify the amount of proteins adsorbed on the sample surfaces. Results indicated that the increase of nanoscale surface roughness caused a significant increase of the amount of adsorbed proteins with a maximum value occurred at 1h (Fig. 3). The increased protein adsorption on the nano-PDMS in the first several minutes could in part be responsible for the antibacterial properties. Conclusions: The relationship between the nanosurface roughness, protein adsorption and bacteria activities was investigated in this study. Data showed that the increased nanoscale surface roughness could increase the amount of proteins adsorbed, specifically, casein, inhibiting both bacteria adhesion and growth significantly while remaining non-toxic to mammalian cells. These results together indicated that the present biomimetic nano-patterned PDMS surface (without any chemical or antibiotic modification) should strongly be considered for reducing infections in catheters and other implanted biomaterials.

Understanding the Role of Polymer Surface Nanoscale Topography on Inhibiting Bacteria Adhesion and Growth.

Catheter-associated infections, most of which are caused by microbial biofilms, are still a serious issue in healthcare and are associated with significant morbidity, mortality, and excessive medical costs. Currently, the use of nanostructured materials, especially materials with nanofeatured topographies, which have more surface area, altered surface energy, enhanced select protein adsorption, and selectively increased desirable cell functions while simultaneously decreasing competitive cell functions, seem to be among the most promising ways for reducing initial bacteria attachment, biofilm formation, and infections. In this study, polydimethylsiloxane (PDMS), a commonly used polymeric catheter material, was formulated to mimic the nanopatterned topography of natural tissue by using a template method with nanotubular anodized titanium. Results showed that increased PDMS surface nanoscale roughness alone can inhibit both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria adhesion and growth for up to 2 days, the time length of the current study. Additionally, increased fibroblast and endothelial cell adhesion on nano-PDMS indicated that this nanoscale topography had no toxic effects toward mammalian cells. Mechanistically, this study also developed a model for the first time to correlate bacteria responses to nanoscale roughness with initial protein and biomolecule adsorption (specifically, casein protein and glucose, which are unique biomolecules that mediate bacteria functions). Data revealed that the increase in nanoscale roughness and associated energy contributed to greater select casein adsorption during the first several minutes of culture, which is critical for decreasing bacteria attachment and growth. In contrast, no significant differences for glucose adsorption between samples before and after nanofabrication were identified. These results together indicated that the present biomimetic nanopatterned PDMS surface without any chemical or antibiotic modification has the potential to combat catheter-associated infections and should be further investigated.

Real time macrophage migration analysis and associated pro-inflammatory cytokine release on transparent carbon nanotube/polymer composite nano-film

Surface chemistry and nanoscale surface morphology are both influential factors for cell adhesion, growth, and differentiation. In particular, cell migration is one of the major markers of initial immune response activation to implanted biomaterials. Despite their indication, it has been difficult to directly examine macrophages on nanoscale materials, because most nanomaterials possess greater thicknesses than nanoscale. This study developed transparent films comprising a carbon nanotube and polymer composite with controlled surface stiffness and nanoscale roughness. As nanoscale surface topography can incite immune cell activation, analysis of the real-time cell migration (including velocity) of macrophages due to changes in nanoscale surface topography of a biopolymer can support the direct relationship between initial macrophage dynamics and corresponding pro-inflammatory responses. Through real-time analysis, we have identified that surface chemistry and surface nanoscale topography are both independent factors mediating macrophage interactions, and, thus, immune cell behavior can be further controlled by the systematic variation of nanoscale surface topography for a given surface chemistry. Considering that the initial immune response can determine the fate and lifetime of implanted biomaterials, this study presents the direct relationship between initial macrophage dynamics and subsequent inflammatory cytokine release on transparent carbon nanotube polymer composites.

Field limit and nano-scale surface topography of superconducting radio-frequency cavity made of extreme type II superconductor

The field limit of superconducting radio-frequency cavity made of type II superconductor with a large Ginzburg-Landau parameter is studied with taking effects of nano-scale surface topography into account. If the surface is ideally flat, the field limit is imposed by the superheating field. On the surface of cavity, however, nano-defects almost continuously distribute and suppress the superheating field everywhere. The field limit is imposed by an effective superheating field given by the product of the superheating field for ideal flat surface and a suppression factor that contains effects of nano-defects. A nano-defect is modeled by a triangular groove with a depth smaller than the penetration depth. An analytical formula for the suppression factor of bulk and multilayer superconductors are derived in the framework of the London theory. As an immediate application, the suppression factor of the dirty Nb processed by the electropolishing is evaluated by using results of surface topographic study. The estimated field limit is consistent with the present record field of nitrogen-doped Nb cavities. Suppression factors of surfaces of other bulk and multilayer superconductors, and those after various surface processing technologies can also be evaluated by using the formula.

Human mesenchymal stem cell response to poly(ε-caprolactone/poly(methyl methacrylate) demixed thin films.

Advances in material sciences have enabled the fabrication of biomaterials which are able to provide the requisite cues to stimulate cells to behave in a specific way. Nanoscale surface topographies are well known to be able to positively influence cell-substrate interactions. This study reports on a novel series of poly(ε-caprolactone) PCL and poly(methyl methacrylate) demixed nanotopographic films as non-biological cell-stimulating cues. The topographic features observed ranged from nanoislands to nanopits. PMMA was observed to segregate to the air interface, while PCL preferred the substrate interface. Preliminary response of human mesenchymal stem cells to these surfaces indicated that the substrate with nanoisland topography has the potential to differentiate to osteogenic, chondrogenic and adipogenic lineages.

Stem cell behavior on tailored porous oxide surface coatings

Nanoscale surface topographies are known to have a profound influence on cell behavior, including cell guidance, migration, morphology, proliferation, and differentiation. In this study, we have observed the behavior of human mesenchymal stem cells cultured on a range of tailored porous SiO2 and TiO2 nanostructured surface coatings fabricated via glancing angle electron-beam deposition. By controlling the physical vapor deposition angle during fabrication, we could control systematically the deposited coating porosity, along with associated topographic features. Immunocytochemistry and image analysis quantitatively revealed the number of adherent cells, as well as their basic cellular morphology, on these surfaces. Signaling pathway studies showed that even with subtle changes in nanoscale surface structures, the behavior of mesenchymal stem cells was strongly influenced by the precise surface structures of these porous coatings.

Biomaterial nanotopography-mediated cell responses: experiment and modeling

The rapid development of fabrication and processing technologies in the past two decades has enabled researchers to introduce nanoscale features into materials which, interestingly, have been shown to greatly regulate the behavior and fate of biological cells. In particular, important cell responses (such as adhesion, proliferation, differentiation, migration, and filopodial growth) have all been correlated with material nanotopography. Given its great potential, intensive efforts have been made, both experimentally and theoretically, to understand why and how cells respond to nanoscale surface features, and this article reviews recent progress in this field. Specifically, a brief overview on the fabrication and modification techniques to create nanotopography on different materials is given first. After that, a summary of important experimental findings on the mediation of nanoscale surface topography on the behavior of various cells, as well as the underlying mechanism, is provided. Finally, both classical and recently developed approaches for modeling nanotopography-mediated cell adhesion and filopodial growth are reviewed.

A Cahn-Hilliard Modeling of Metal Oxide Thin Films for Advanced CMP Applications

Chemical mechanical planarization (CMP) process enables topographic selectivity through formation of a protective oxide thin film on the recessed locations of the deposited metal layer, while a continuous chemical oxidation reaction is followed by mechanical abrasion takes place on the protruding locations. This paper demonstrates a new approach to CMP process optimization in terms of analyzing the nano-scale surface topography of the protective metal oxide films and modeling their growth through a Cahn-Hilliard equation (CHE) approach as an alternative to classical nucleation theory. It is observed that the material removal rate mechanisms and the consequent planarization performance depend on the nature of nucleation of the metal oxide films, which is a function of the oxidizer concentration of the CMP slurry.

Super-hydrophilic nanostructured titanium surface improves implant osseointegration by mediating macrophage polarization (CAM5P.233)

Abstract Aims: The purpose of this study was to fabricate a nanostructured surface on a titanium (Ti) implant that can regulate the macrophage inflammatory response and thereby improve the performance of the implant. Materials and Methods: Pure Ti samples were anodized and then subjected to UV irradiation to generate super-hydrophilic, nanostructured TiO2 surfaces. Their surface characteristics were analyzed, and osteointegration as well as the inflammatory response of modified implants were evaluated in vivo and in vitro. Results: Both nanostructured TiO2 surfaces showed super-hydrophilic and multi-tubular surface topography with diameters of 30 nm (NT5) and 80 nm (NT20). Although the in vitro osteogenic activity of bMSCs was similar on the two surfaces, the NT5 surface showed more bone formation, less inflammation, and a reduced CD68+ macrophage distribution in vivo compared to the NT20 and polished Ti surfaces. Further experiments revealed that the NT5 surfaces suppressed macrophage secretion of inflammatory cytokines and induced M2 polarization, which is associated with improved healing. By contrast, promoted M1 polarization could further impair bone regeneration. Conclusion: The results demonstrate the dominant role of macrophage-related inflammation in bone healing around implants and that nano-scale surface topography may have an immune-regulating effect that can support the success of implants.

Nanoscale Surface Topography Reshapes Neuronal Growth in Culture

Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nanopillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nanopillars with a typical distance between adhesion points of about 500 nm. These neurons produce fewer neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted with a differential surface topography, neurons specify an axon preferentially on nanopillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.

Bone marrow stromal cell adhesion and morphology on micro- and sub-micropatterned titanium.

The objective of this study was to investigate the adhesion and morphology of bone marrow derived stromal cells (BMSCs) on bulk titanium (Ti) substrates with precisely-patterned surfaces consisting of groove-based gratings with groove widths ranging from 50 micro m down to 0.5 micro m (500 nm). Although it is well known that certain surface patterning enhances osteoblast (bone-forming cell) functions, past studies on cell-pattern interactions reported in the literature have heavily relied on surface patterning on materials with limited clinical relevance for orthopedic applications, such as polymeric substrates. The clinical need for improving osseointegration and juxtaposed bone formation around load-bearing Ti implants motivated this in vitro study. BMSCs were selected as model cells due to their important role in bone regeneration. The results showed significantly greater BMSC adhesion density and more favorable cell morphology on sub-micropatterned gratings when compared with larger micropatterned gratings and non-patterned control surfaces after both 24 hr and 72 hr cultures. We observed increasing cellular alignment and elongation with decreasing feature size. We also identified two distinctive cellular morphologies: Type I-Attached and spread cells that elongated along the pattern axes; and Type II-Superficially adhered round cells. Sub-micropatterned gratings demonstrated significantly greater Type I cell density than the non-patterned control, and lower Type II cell density than the larger micropatterned gratings. Collectively, these results suggest potential for rationally designing nano-scale surface topography on Ti implants to improve osseointegration.

Antimicrobial oligo(p-phenylene-ethynylene) film deposited by resonant infrared matrix-assisted pulsed laser evaporation

The antimicrobial oligomer, oligo(p-phenylene-ethynylene) (OPE), was deposited as thin films by resonant infrared matrix-assisted pulsed laser evaporation (RIR-MAPLE) on solid substrates and exhibited light-induced biocidal activity. The biocidal activity of OPE thin films deposited by spin-coating and drop-casting was also investigated for comparison. Enhanced bacterial attachment and biocidal efficiency of the film deposited by RIR-MAPLE were observed and attributed to nanoscale surface topography of the thin film.

Protein Immobilization in Hollow Nanostructures and Investigation of the Adsorbed Protein Behavior

Understanding nanomaterial-biomolecule interactions is critical to develop broad applications in sensors, devices, and therapeutics. During the past decade, in-depth studies have been performed on the effect of nanoscale surface topography on adsorbed protein structure and function. However, a fundamental understanding of nanobio interactions at concave surfaces is limited; the greatest challenge is to create a nanostructure that allows such interactions to occur and to be characterized. We have synthesized hollow nanocages (AuNG) through careful control of morphology and surface chemistry. Lysozyme was used as a model to probe interactions between a protein and these nanostructures. Solid Au nanoparticles with a similar morphology and surface chemistry were also used as a reference. Through a series of quantitative analyses of protein adsorption profiles and enzymatic activity assays of both nanobioconjugates, we discovered that a significant amount of protein could be delivered into the core of AuNG, while maintaining a substantial fraction of native activity.

Weathering by tree-root-associating fungi diminishes under simulated Cenozoic atmospheric CO<sub>2</sub> decline

Abstract. Trees dominate terrestrial biotic weathering of silicate minerals by converting solar energy into chemical energy that fuels roots and their ubiquitous nutrient-mobilising fungal symbionts. These biological activities regulate atmospheric CO2 concentrations ([CO2]a) over geologic timescales by driving calcium and magnesium fluvial ion export and marine carbonate formation. However, the important stabilising feedbacks between [CO2]a and biotic weathering anticipated by geochemical carbon cycle models remain untested. We report experimental evidence for a negative feedback across a declining Cenozoic [CO2]a range from 1500 to 200 ppm, whereby low [CO2]a curtails mineral surface alteration via trenching and etch pitting by arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungal partners of tree roots. Optical profile imaging using vertical scanning interferometry reveals changes in nanoscale surface topography consistent with a dual mode of attack involving delamination and trenching by AM and EM fungal hyphae on phyllosilicate mineral flakes. This is consistent with field observations of micropores in feldspar, hornblende and basalt, purportedly caused by EM fungi, but with little confirmatory evidence. Integrating these findings into a process-based biotic weathering model revealed that low [CO2]a effectively acts as a "carbon starvation" brake, causing a three-fold drop in tree-driven fungal weathering fluxes of calcium and magnesium from silicate rock grains as [CO2]a falls from 1500 to 200 ppm. The feedback is regulated through the action of low [CO2]a on host tree productivity and provides empirical evidence for the role of [CO2]a starvation in diminishing the contribution of trees and mycorrhizal fungi to rates of biological weathering. More broadly, diminished tree-driven weathering under declining [CO2]a may provide an important contributory mechanism stabilising Earth's [CO2]a minimum over the past 24 million years.

Pattern formation on Ge by low energy ion beam erosion

Modification of nanoscale surface topography is inherent to low-energy ion beam erosion processes and is one of the most important fields of nanotechnology. In this report a comprehensive study of surface smoothing and self-organized pattern formation on Ge(100) by using different noble gases ion beam erosion is presented. The investigations focus on low ion energies (⩽ 2000 eV) and include the entire range of ion incidence angles. It is found that for ions (Ne, Ar) with masses lower than the mass of the Ge target atoms, no pattern formation occurs and surface smoothing is observed for all angles of ion incidence. In contrast, for erosion with higher mass ions (Kr, Xe), ripple formation starts at incidence angles of about 65° depending on ion energy. At smaller incident angles surface smoothing occurs again. Investigations of the surface dynamics for specific ion incidence angles by changing the ion fluence over two orders of magnitude gives a clear evidence for coarsening and faceting of the surface pattern. Both observations indicate that gradient-dependent sputtering and reflection of primary ions play crucial role in the pattern evolution, just at the lowest accessible fluences. The results are discussed in relation to recently proposed redistributive or stress-induced models for pattern formation. In addition, it is argued that a large angular variation of the sputter yield and reflected primary ions can significantly contribute to pattern formation and evolution as nonlinear and non-local processes as supported by simulation of sputtering and ion reflection.

Fabrication of Nanostructured Hierarchical Coatings Composed of Calcium Phosphate/Titanate on Titanium Substrate

Titanium (Ti) and its alloys have been widely used in orthopedic and dental applications. The bonding between Ti implants and tissues is significantly important in the clinical applications, which is highly relative to the characteristics of implant surface with surface improvement by various coatings such as hydroxyapatite (HA). Meanwhile, the characteristics of modified implants surface can induce special biological response of osteogenic cells to improve the bone-implant bonding. In this paper, in order to improve Ti bioactivity and enhance its osteointegration, calcium phosphate (CaP)/titanate coatings were prepared on Ti with nanoscale surface topographies by a two-step hydrothermal process. Firstly, titanium meshes were treated in sodium hydroxide solutions at two temperatures (200 °C or 240 °C). Then, the treated meshes were immersed in calcium phosphate solutions containing various concentrations of cyclohexane-hexacarboxylic acid (H6L) under hydrothermal conditions. After this two-step process, calcium phosphate deposits, confirmed to be apatite/titanate by energy dispersive X-ray spectroscopy, were successfully formed on the treated Ti mesh surface. These results suggested that this two-step hydrothermal method can be successfully applied to fabricate robust and hierarchical nanostructured coatings of CaP/titanate on Ti implants.

Addition of nanoscaledbioinspiredsurface features: A revolution for bone related implants and scaffolds?

Our expanding ability to handle the "literally invisible" building blocks of our world has started to provoke a seismic shift on the technology, environment and health sectors of our society. During the last two decades, it has become increasingly evident that the "nano-sized" subunits composing many materials—living, natural and synthetic—are becoming more and more accessible for predefined manipulations at the nanosize scale. The use of equally nanoscale sized or functionalised tools may, therefore, grant us unprecedented prospects to achieve many therapeutic aims. In the past decade it became clear that nano-scale surface topography significantly influences cell behaviour and may, potentially, be utilised as a powerful tool to enhance the bioactivity and/ or integration of implanted devices. In this review, we briefly outline the state of the art and some of the current approaches and concepts for the future utilisation of nanotechnology to create biomimetic implantable medical devices and scaffolds for in vivo and in vitro tissue engineering,with a focus on bone. Based on current knowledge it must be concluded that not the materials and surfaces themselves but the systematic biological evaluation of these new material concepts represent the bottleneck for new biomedical product development based on nanotechnological principles.

Dynamic and reversible surface topography influences cell morphology

Microscale and nanoscale surface topography changes can influence cell functions, including morphology. Although in vitro responses to static topography are novel, cells in vivo constantly remodel topography. To better understand how cells respond to changes in topography over time, we developed a soft polyacrylamide hydrogel with magnetic nickel microwires randomly oriented in the surface of the material. Varying the magnetic field around the microwires reversibly induced their alignment with the direction of the field, causing the smooth hydrogel surface to develop small wrinkles; changes in surface roughness, ΔRRMS , ranged from 0.05 to 0.70 μm and could be oscillated without hydrogel creep. Vascular smooth muscle cell morphology was assessed when exposed to acute and dynamic topography changes. Area and shape changes occurred when an acute topographical change was imposed for substrates exceeding roughness of 0.2 μm, but longer-term oscillating topography did not produce significant changes in morphology irrespective of wire stiffness. These data imply that cells may be able to use topography changes to transmit signals as they respond immediately to changes in roughness.

Nanoscale surface pattern formation kinetics on germanium irradiated by Kr+ions

Nanoscale surface topography evolution on Ge surfaces irradiated by 1 keV Kr+ ions is examined in both directions perpendicular and parallel to the projection of the ion beam on the surface. Grazing incidence small angle x-ray scattering is used to measure in situ the evolution of surface morphology via the linear dispersion relation. A transition from smoothing (stability) to pattern-forming instability is observed at a critical ion incidence angle of approximately 62◦ with respect to the surface normal. The linear theory quadratic coefficients which determine the surface stability/instability are determined as a function of bombardment angle. The Ge surface evolution during Kr+ irradiation is qualitatively similar to that observed for Ar+ irradiation of Si. However, in contrast to the case of Si under Ar+ irradiation, the critical angle separating stability and instability for Ge under Kr+ irradiation cannot be quantitatively reproduced by the simple Carter-Vishnyakov mass redistribution model.

Photopolymerized microfeatures for directed spiral ganglion neurite and Schwann cell growth

Cochlear implants (CIs) provide auditory perception to individuals with severe hearing impairment. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by electrical current spread in the inner ear. Directing nerve cell processes towards target electrodes may reduce the problematic current spread and improve stimulatory specificity. In this work, photopolymerization was used to fabricate micro- and nano-patterned methacrylate polymers to probe the extent of spiral ganglion neuron (SGN) neurite and Schwann cell (SGSC) contact guidance based on variations in substrate topographical cues. Micropatterned substrates are formed in a rapid, single-step reaction by selectively blocking light with photomasks which have parallel line-space gratings with periodicities of 10–100 μm. Channel amplitudes of 250 nm–10 μm are generated by modulating UV exposure time, light intensity, and photoinitiator concentration. Gradual transitions are observed between ridges and grooves using scanning electron and atomic force microscopy. The transitions stand in contrast to vertical features generated via etching lithographic techniques. Alignment of neural elements increases significantly with increasing feature amplitude and constant periodicity, as well as with decreasing periodicity and constant amplitude. SGN neurite alignment strongly correlates (r = 0.93) with maximum feature slope. Multiple neuronal and glial types orient to the patterns with varying degrees of alignment. This work presents a method to fabricate gradually-sloping micropatterns for cellular contact guidance studies and demonstrates spatial control of inner ear neural elements in response to micro- and nano-scale surface topography.