Hydroxyapatite Pellets as Versatile Model Surfaces for Systematic Adhesion Studies on Enamel: A Force Spectroscopy Case Study.

Research into materials for medical application draws inspiration from naturally occurring or synthesized surfaces, just like many other research directions. For medical application of materials, particular attention has to be paid to biocompatibility, osseointegration, and bacterial adhesion behavior. To understand their properties and behavior, experimental studies with natural materials such as teeth are strongly required. The results, however, may be highly case-dependent because natural surfaces have the disadvantage of being subject to wide variations, for instance in their chemical composition, structure, morphology, roughness, and porosity. A synthetic surface which mimics enamel in its performance with respect to bacterial adhesion and biocompatibility would, therefore, facilitate systematic studies much better. In this study, we discuss the possibility of using hydroxyapatite (HAp) pellets to simulate the surfaces of teeth and show the possibility and limitations of using a model surface. We performed single-cell force spectroscopy with single Staphylococcus aureus cells to measure adhesion-related parameters such as adhesion force and rupture length of cell wall proteins binding to HAp and enamel. We also examine the influence of blood plasma and saliva on the adhesion properties of S. aureus. The results of these measurements are matched to water wettability, elemental composition of the samples, and the change in the macromolecules adsorbed over time on the surface. We found that the adhesion properties of S. aureus were similar on HAp and enamel samples under all conditions: Significant decreases in adhesion strength were found equally in the presence of saliva or blood plasma on both surfaces. We therefore conclude that HAp pellets are a good alternative for natural dental material. This is especially true when slight variations in the physicochemical properties of the natural materials may affect the experimental series.

Strength of bacterial adhesion on nanostructured surfaces quantified by substrate morphometry.

Microbial adhesion and the subsequent formation of resilient biofilms at surfaces are decisively influenced by substrate properties, such as the topography. To date, studies that quantitatively link surface topography and bacterial adhesion are scarce, as both are not straightforward to quantify. To fill this gap, surface morphometry combined with single-cell force spectroscopy was performed on surfaces with irregular topographies on the nano-scale. As surfaces, hydrophobized silicon wafers were used that were etched to exhibit surface structures in the same size range as the bacterial cell wall molecules. The surface structures were characterized by a detailed morphometric analysis based on Minkowski functionals revealing both qualitatively similar features and quantitatively different extensions. We find that as the size of the nanostructures increases, the adhesion forces decrease in a way that can be quantified by the area of the surface that is available for the tethering of cell wall molecules. In addition, we observe a bactericidal effect, which is more pronounced on substrates with taller structures but does not influence adhesion. Our results can be used for a targeted development of 3D-structured materials for/against bio-adhesion. Moreover, the morphometric analysis can serve as a future gold standard for characterizing a broad spectrum of material structures.

Effect of Fluoride Treatment on the Acid Resistance of Hydroxyapatite.

The etching behavior of polycrystalline synthetic hydroxyapatite samples has been evaluated to explore the protective impact of fluoride on a tooth-like model system. Etching rates before and after fluoridation with a NaF solution at pH 6 were determined by atomic force microscopy. Despite a very low F concentration of ca. 0.2 atom % in the hydroxyapatite surface, a very strong effect on the acid resistance can be observed. Depending on the crystal orientation, etching in a NaAc buffer at pH 4.5 was completely inhibited for at least 5 min. The major part of the surface withstood etching even for more than 23 min. These results give new insights into how the amount of incorporated fluoride in hydroxyapatite correlates with its protective impact.

Photoactivatable Mussel-Based Underwater Adhesive Proteins by an Expanded Genetic Code.

Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.

Enhanced adhesion of Streptococcus mutans to hydroxyapatite after exposure to saliva.

Streptococcus mutans cells form robust biofilms on human teeth and are strongly related to caries incidents. Hence, understanding the adhesion of S. mutans in the human oral cavity is of major interest for preventive dentistry. In this study, we report on atomic force microscopy-based single-cell force spectroscopy measurements of S. mutans cells to hydroxyapatite surfaces. We observe for almost all measurements a significant difference in adhesion strength for S. mutans as well as for Staphylococcus carnosus cells. However, the increase in adhesion strength after saliva exposure is much higher for S. mutans cells compared to S. carnosus cells. Our results demonstrate that S. mutans cells are well adapted to their natural environment, the oral cavity. This ability promotes the biofilm-forming capability of that species and hence the production of caries-provoking acids. In consequence, understanding the fundamentals of this mechanism may pave a way towards more effective caries-reducing techniques.

Time Dependence of Fluoride Uptake in Hydroxyapatite.

Fluoridation of enamel is believed to provide an effective tool to protect teeth from caries, but there is still little information on the time scale of fluoride uptake. In this study, highly compressed pellets of hydroxyapatite are used as first-order model systems to approximate the mineral component of natural enamel for investigations on the time-dependence of fluoride uptake. We found that both the overall amount of fluoride as well as the mean thickness of the fluoridated surface layer cannot be extended to any values just by increasing the application time of a fluoride containing agent. Instead, both parameters start to become constant on a time scale of about 3 min. The present results as obtained on a synthetic model "tooth" show that the time scale to provide the maximum amount of fluoride possible is of the same order of magnitude as that in usual daily practice in dental care when applying toothpastes or mouth rinses.

Synthesis of Hydroxyapatite Substrates: Bridging the Gap between Model Surfaces and Enamel.

Hydroxyapatite substrates are common biomaterials, yet samples of natural teeth do not meet the demands for well-defined, highly reproducible properties. Pellets of hydroxyapatite were produced via the field assisted sintering technology (FAST) as well as via pressureless sintering (PLS). The applied synthesis routes provide samples of very high density (95%-99% of the crystallographic density) and of very low surface roughness (lower than 1 nm when averaged per 1 µm2). The chemical composition of the raw material (commercial HAP powder) as well as the crystalline structure is maintained by the sintering processes. These specimens can therefore be considered as promising model surfaces for studies on the interactions of biomaterial with surfaces of biological relevance, as demonstrated for the adsorption of BSA proteins.

Is adhesion superficial? Silicon wafers as a model system to study van der Waals interactions.

Adhesion is a key issue for researchers of various fields, it is therefore of uppermost importance to understand the parameters that are involved. Commonly, only surface parameters are employed to determine the adhesive forces between materials. Yet, van der Waals forces act not only between atoms in the vicinity of the surface, but also between atoms in the bulk material. In this review, we describe the principles of van der Waals interactions and outline experimental and theoretical studies investigating the influence of the subsurface material on adhesion. In addition, we present a collection of data indicating that silicon wafers with native oxide layers are a good model substrate to study van der Waals interactions with coated materials.