Li-Chong Xu

Effects of toxic metals and chemicals on biofilm and biocorrosion

Microbes in marine biofilms aggregated into clusters and increased the production of extracellular polymeric substances (EPS), by over 100% in some cases, when the seawater media containing toxic metals and chemicals, such as Cd(II), Cu(II), Pb(II), Zn(II), AI(III), Cr(III), glutaraldehyde, and phenol. The formation of microbial cluster and the increased production of EPS, which contained 84-92% proteins and 8-16% polysaccharides, accelerated the corrosion of the mild steel. However, there was no quantitative relationship between the degree of increased corrosion and the toxicity of metals/chemicals towards sulfate-reducing bacteria, or the increased EPS production.

Quantification of bacterial adhesion forces using atomic force microscopy (AFM).

This study demonstrated that atomic force microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria, and to quantify the tip-cell interaction force and the surface elasticity. Results show that the adhesion force between the Si3N4 tip and the bacteria surface was in the range from -3.9 to -4.3 nN. On the other hand, the adhesion forces at the periphery of the cell-substratum contact surface ranged from -5.1 to -5.9 nN and those at the cell-cell interface ranged from -6.5 to -6.8 nN. The two latter forces were considerably greater than the former one, most likely due to the accumulation of extracellular polymer substance (EPS). Results also show that the elasticity varied on the cell surface.

Submicron-textured biomaterial surface reduces staphylococcal bacterial adhesion and biofilm formation

Staphylococci are among the most important pathogens causing bloodstream infections associated with implanted medical devices. Control of bacterial adhesion to material surfaces is important for prevention of biofilm formation and biomaterial-associated infections. In this study, we hypothesized that submicron (staphylococcal bacterial dimension) surface textures may reduce the bacterial adhesion via a decrease in surface area that bacteria can contact, and subsequently inhibit biofilm formation. Poly(urethane urea) films were textured with two different sizes of submicron pillars via a two-stage replication process. Adhesion of two bacterial strains (Staphylococcus epidermidis RP62A and S. aureus Newman) was assessed over a shear stress range of 0-13.2 dyn cm(-2) using a rotating disk system in physiological buffer solutions. Significant decreases in bacterial adhesion were observed on textured surfaces for both strains compared with smooth controls. Biofilm formation was further tested on surfaces incubated in solution for either 2 or 5 days and it was found that biofilm formation was dramatically inhibited on textured surfaces. The results of the approaches used in this work demonstrate that patterned surface texturing of biomaterials provides an effective means to reduce staphylococcal adhesion and biofilm formation on biomaterial surfaces, and thus to prevent biomaterial-associated infections.

Proteins, platelets, and blood coagulation at biomaterial interfaces

Blood coagulation and platelet adhesion remain major impediments to the use of biomaterials in implantable medical devices. There is still significant controversy and question in the field regarding the role that surfaces play in this process. This manuscript addresses this topic area and reports on state of the art in the field. Particular emphasis is placed on the subject of surface engineering and surface measurements that allow for control and observation of surface-mediated biological responses in blood and test solutions. Appropriate use of surface texturing and chemical patterning methodologies allow for reduction of both blood coagulation and platelet adhesion, and new methods of surface interrogation at high resolution allow for measurement of the relevant biological factors.

Application of atomic force microscopy in the study of microbiologically influenced corrosion

Abstract This paper demonstrates the use of the atomic force microscope in high-resolution topographical imaging of bacteria, biofilm, and corroded steel surfaces, and in the quantification of localized corrosion. The nanometric physicochemical and mechanical properties of a single cell and bacterial biofilm surface are characterized by force mapping. The corrosion results in two different sulfate-reducing bacteria cultures showed that the patterns of pitting and the degree of corrosion of mild steel were related to the bacterial isolates. Results from measurement of the tip–biofilm and the tip–cell adhesion forces indicated that the extracellular polymeric substances were mainly distributed in the cell–substratum periphery or the cell–cell interface in the biofilm.

Atomic Force Microscopy Studies of the Initial Interactions Between Fibrinogen and Surfaces

Atomic force microscopy (AFM) was used to analyze the interactions between fibrinogen and model surfaces having different levels of water wettability. In contrast to most AFM studies, proteins were coupled to the substrate while model surface colloids were attached to the end of the AFM probe, thereby ensuring that proteins undergo only a single compression/decompression cycle. Similar values of adhesion force were observed between fibrinogen and all of the highly wettable surfaces; in the same manner, fibrinogen showed similar adhesion forces against all poorly wettable surfaces, with a step-like transition observed between the two groups. Relationships between the adhesion forces and loading rates were used to analyze the energy profiles involved in protein/surface interactions. Multiple energy barriers were found in the interaction of proteins with poorly wettable surfaces; whereas a single energy barrier was found for protein interactions with highly wettable surfaces. Contact time-dependent adhesion data were fit to an exponential model and showed that the rate constants of the protein unfolding process on highly wettable surface were smaller at low loading rates, but increased rapidly to yield values similar to those on poorly wettable surfaces at high loading rates. The activation energies of protein unfolding derived from the data offer insight into the role of surface wettability in affecting adhesion, conformational changes, and ultimately, the activity of proteins at biomaterial surfaces.

Atomic Force Microscopy Study of Microbiologically Influenced Corrosion of Mild Steel

The anaerobic corrosion of mild steel in seawater was studied by atomic force microscopy (AFM). In the presence of sulfate reducing bacteria (SRB), corrosion was intensified and accelerated. A biofilm consists of heterogeneous microbial cells and extracellular polymeric substance with interstitial voids was observed on the surface of mild steel coupons. The greatest damage of steel occurred beneath the biofilm, in the form of pitting corrosion. The corrosion of steel can be quantified through section and bearing analyses. The depth of pits increased linearly with time whereas the volume of pits increased as t{sup 2.83}, the 2.83 power of time. Compared with a control experiment without SRB, the depth of pitting corrosion is about one order of magnitude higher. Weight loss estimates from AFM images are about one order of magnitude smaller than actual weight loss experimental results. The problems of AFM quantification of corrosion rate at extended stage of corrosion are discussed.

Substrate curvature sensing through Myosin IIa upregulates early osteogenesis

Topographical cues mimicking the extracellular matrix (ECM) have demonstrated control over a diverse range of cellular behaviours including: initial adhesion, migration, cell growth, differentiation and death. How cells sense, and in turn translate, the topographical cues remains to be answered, but likely involves interactions through interfacial forces that influence cytoskeletal structure and integrin clustering, leading to the downstream activity of intracellular signalling cascades. Electrospun fibers have shown significant success as a biomimetic topography for bone tissue engineering applications, but mechanisms by which osteoprogenitor cells translate the fiber geometry into intracellular signalling activity is only recently being examined. We hypothesized that increased cellular differentiation observed on fibrous topography is due to acto-myosin contractility and cellular stiffness via the small GTPase RhoA. In order to evaluate this hypothesis, MC3T3-E1 osteoprogenitor cells were grown on poly(methyl methacrylate) (PMMA) fibers of 1.153 ± 0.310 μm diameter. The elastic modulus of the cell surface was measured by atomic force microscopy (AFM) with a colloidal probe. Overall cellular stiffness was found to increase more than three-fold in osteoprogenitors adhered to a fiber, as opposed to those grown on a flat substrate. Pharmacological inhibition of RhoA signalling activity decreased cellular stiffness and cytoskeletal integrity of osteoprogenitors growing on fibrous substrates. Finally, we demonstrated not only RhoA activity through its effector Rho-associated coiled coil kinase II (ROCKII), but also Myosin IIa promotes early osteogenic differentiation, as shown by alkaline phosphatase (ALP) staining. Previous studies have demonstrated the importance of ROCKII on early differentiation. Our results shed light on mechanisms underlying geometry sensing by highlighting the role of Myosin IIa in addition to ROCKII and could ultimately contribute to scaffold design strategies.

Dynamics of hydrated polyurethane biomaterials: Surface microphase restructuring, protein activity and platelet adhesion

Microphase separation is a central feature of segmented polyurethane biomaterials and contributes to the biological response to these materials. In this study we utilized atomic force microscopy (AFM) to study the dynamic restructuring of three polyurethanes having soft segment chemistries of interest in biomedical applications. For each of the materials we followed the changes in near surface mechanical properties during hydration, as well as fibrinogen activity and platelet adhesion on these surfaces. Both AFM phase imaging and force mode analysis demonstrated that these polyurethane biomaterials underwent reorientation and rearrangement resulting in a net enrichment of hard domains at the surface. Fibrinogen activity and platelet adhesion on the polyurethane surfaces were found to decrease with increasing hydration time. The findings suggest that water-induced enrichment of hydrophilic hard domains at the surface changes the local surface physical and chemical properties in a way that influences the conformation of fibrinogen, changing the availability of the platelet-binding sites in the protein. This work demonstrates that the hydrated polyurethane biomaterial interface is a complex and dynamic environment where the surface chemistry is changing, altering the activity of fibrinogen and affecting blood platelet adhesion.

Microphase separation structure influences protein interactions with poly(urethane urea) surfaces

Microphase separation is an important characteristic of polyurethane copolymer biomaterials. An improved understanding of the effects of microphase structure on protein interactions with the polymeric biomaterial surface is essential for the development and application of new biomaterials intended for implantation into the body. In this study, an array of atomic force microscopy (AFM) techniques were used to visualize the phase separation structure in a hydrated poly(urethane urea) (PUU) material and to correlate that structure with molecular interactions at the molecular level. Sequential in situ AFM phase images showed that the hard domains present a dynamic environment and undergo rearrangement and enrichment at the surface when hydrated. Adhesion forces measured using a protein-modified AFM probe suggests that the PUU surface became less adhesive to protein with hydration time, consistent with other physical characterizations. Force measurements were used to quantify and correlate mechanical properties and local adhesion forces for bovine serum albumin, and results showed that low adhesion forces were primarily associated with polar hard domain regions. A nanogold-labeled protein conjugate was used to visualize individual protein adsorption to the separate microstructures on the PUU surface, with preferential protein adsorption seen on the more apolar hydrophobic soft segment regions. Together, the results suggest that the microphase separation structure mediates local surface microenvironments that influence biological interactions with the surface.