Combining QCM-D with live-cell imaging reveals the impact of serum proteins on the dynamics of fibroblast adhesion on tannic acid-functionalised surfaces.

Nanocoatings based on plant polyphenols have been recently suggested as a potent strategy for modification of implant surfaces for enhancing host cell attachment and reducing bacterial colonisation. In this study we aimed to investigate how serum proteins impact the early adhesion dynamics of human gingival fibroblasts onto titanium surfaces coated with tannic acid (TA). Silicate-TA nanocoatings were formed on titanium and pre-conditioned in medium supplemented with 0, 0.1, 1 or 10% FBS for 1 hour. Dynamics of fibroblasts adhesion was studied using quartz crystal microbalance with dissipation (QCM-D). Time-lapse imaging was employed to assess cell area and motility, while immunofluorescence microscopy was used to examine cell morphology and focal adhesion formation. Our results showed that in serum-free medium, fibroblasts demonstrated enhanced and faster adhesion to TA coatings compared to uncoated titanium. Increasing the serum concentration reduced cell adhesion to nanocoatings, resulting in nearly complete inhibition at 10% FBS. This inhibition was not observed for uncoated titanium at 10% FBS, although cell adhesion was delayed and progressed slower compared to serum-free conditions. In addition, 1% FBS dramatically reduced cell adhesion on uncoated titanium. We revealed a positive relationship between changes in dissipation and changes in cell spreading area, and a negative relationship between dissipation and cell motility. In conclusion, our study demonstrated that serum decreases fibroblasts interaction with surfaces coated with TA in a concentration dependent manner. This suggests that controlling serum concentration can be used to regulate or potentially prevent fibroblasts adhesion onto TA-coated titanium surfaces.

Can polyphenolic surface modifications prevent fungal colonization of titanium dental implants?

Oral biofilms can be a major health problem causing infections and chronic inflammation of mucosal tissue. While much effort is put in the investigation of bacteria in biofilms, the role of fungi is often neglected, despite Candida albicans playing a key role in the formation of multispecies oral biofilms. With the rise of antibiotic resistance, new strategies to reduce microbial growth need to be found. Therefore, plant derived polyphenolic molecules have been suggested to reduce both adhesion and growth of pathogenic bacteria and fungi. In this study, we investigated the use of polyphenolic coatings to reduce adhesion and biofilm formation of C. albicans BWP17 on titanium implants. Tannic acid and pyrogallol coatings altered the hydrophobic and charge properties of titanium surfaces, and both compounds were gradually released as active molecules over time. Despite such effects, we found no significant inhibition on growth and biofilm formation of C. Albicans, indicating that the release of active molecules from the coatings did not reach relevant inhibitory concentrations. However, a potential antibiofilm effect was observed by the pH-dependent disassembly of the polyphenolic layer, which caused the biofilm to detach. Hence, further efforts are required to create tailored implant surfaces, which sustainably reduce microbial growth and adhesion.

In Vitro Monitoring of Magnesium-Based Implants Degradation by Surface Analysis and Optical Spectroscopy.

Magnesium (Mg)-based degradable alloys have attracted substantial attention for tissue engineering applications due to their biodegradability and potential for avoiding secondary removal surgeries. However, insufficient data in the existing literature regarding Mg's corrosion and gas formation after implantation have delayed its wide clinical application. Since the surface properties of degradable materials constantly change after contact with body fluid, monitoring the behaviour of Mg in phantoms or buffer solutions could provide some information about its physicochemical surface changes over time. Through surface analysis and spectroscopic analysis, we aimed to investigate the structural and functional properties of degradable disks. Since bubble formation may lead to inflammation and change pH, monitoring components related to acidosis near the cells is essential. To study the bubble formation in cell culture media, we used a newly developed Mg alloy (based on Mg, zinc, and calcium), pure Mg, and commercially available grade 2 Titanium (Ti) disks in Dulbecco's Modified Eagle Medium (DMEM) solution to observe their behaviour over ten days of immersion. Using surface analysis and the information from near-infrared spectroscopy (NIRS), we concluded on the conditions associated with the medical risks of Mg alloy disintegration. NIRS is used to investigate the degradation behaviour of Mg-based disks in the cell culture media, which is correlated with the surface analysis where possible.

Multifunctional Surfaces for Improving Soft Tissue Integration.

Metallic implants are widely used in diverse clinical applications to aid in recovery from lesions or to replace native hard tissues. However, the lack of integration of metallic surfaces with soft tissue interfaces causes the occurrence of biomaterial-associated infections, which can trigger a complicated inflammatory response and, ultimately, implant failure. Here, a multifunctional implant surface showing nanoscale anisotropy, based on the controlled deposition of cellulose nanocrystals (CNC), and biological activity derived from platelet lysate (PL) biomolecules sequestered and presented on CNC surface, is proposed. The anisotropic radial nanopatterns are produced on polished titanium surfaces by spin-coating CNC at high speed. Furthermore, CNC surface chemistry allows to further sequester and form a coating of bioactive molecules derived from PL. The surface anisotropy provided by CNC guides fibroblasts growth and alignment up to 14 days of culture. Moreover, PL-derived biomolecules polarize macrophages toward the M2-like anti-inflammatory phenotype. These results suggest that the developed multifunctional surfaces can promote soft tissue integration to metallic implants and, at the same time, prevent bacterial invasion, tissue inflammation, and failure of biomedical metallic implants.

Peri-implant soft tissue integration in humans - influence of materials: A study protocol for a randomised controlled trial and a pilot study results.

BACKGROUND: Recently, there has been a growing interest in mucointegration as the formation of an early and long-standing soft tissue barrier seems essential for both the initial healing and long-term implant survival. AIM: To develop an experimental method to characterize the mucointegration of different transgingival materials (titanium (Ti), polyetheretherketone (PEEK), polymethylmethacrylate (PMMA), zirconia (Zi), polymer infiltrated ceramic network (PICN), cobalt-chrome (Co-Cr), and lithium disilicate (LD)) in a human model. METHODS: The study is designed as a multi-part randomized controlled clinical trial. Ninety bone level Straumann implants will randomly receive an experimental, custom-made abutment to allow for the removal of the abutment together with the surrounding soft tissues using a punch biopsy device at 8 weeks of healing (10 per material). The specimens will be further processed for non-decalcified histology, followed by histomorphometric analysis. The same protocol will be used for additional 90 implants-abutments, but during harvesting, soft tissues will be separated from the abutment and processed for immunohistochemistry in order to study tissue inflammation and vascularization, while the abutments will undergo SEM analysis. Additionally, in vitro analyses, including SEM and profilometry, will be performed in order to characterize surface topography of all experimental materials. CONCLUSION: The limited number of pilot samples presented herein indicate that the use of custom-made abutments in humans is a reproducible method to study peri-implant soft tissue integration. This further intensifies the rationale to compare different abutment materials, used as transgingival components in daily practice, under the same conditions.

Silicate-Phenolic Networks: Coordination-Mediated Deposition of Bioinspired Tannic Acid Coatings.

Surface modification with polyphenolic molecules has been pursued in biomedical materials owing to their antioxidant, anti-inflammatory, and antimicrobial characteristics. Recently, the use of silicic acid (Siaq ) as a mediator for efficient surface deposition of tannic acid (TA) was reported, but the postulated Si-TA polymeric networks were not characterized. Herein, we present unambiguous evidence for silicate-TA networks that involve Si-O-C motifs by using solid-state NMR spectroscopy, further supported by XPS and ToF-SIMS. By using QCM-D we demonstrate the advantages of Siaq , compared to using transition-metal ions, to improve the coating efficiency under mildly acidic conditions. The presented homogenous coating buildup and validated applicability in inorganic buffers broadens the use of TA for surface modifications in technological and biomedical applications.

Silicic Acid-Mediated Formation of Tannic Acid Nanocoatings.

Tannic acid (TA) adheres to a broad variety of different materials and forms versatile surface coatings for technical and biological applications. In mild alkaline conditions, autoxidation processes occur and a firm monolayer is formed. Up to now, thicker coatings are obtained in only a cross-linked multilayer fashion. This study presents an alternative method to form continuous TA coatings using orthosilicic acid (Siaq). Adsorption kinetics and physical properties of TA coatings in the presence of Siaq were determined using a quartz-crystal microbalance and nanoplasmonic spectroscopy. An in situ TA layer thickness of 200 nm was obtained after 24 h in solutions supplemented with 80 µM Siaq. Dry-state measurements indicated a highly hydrated layer in situ. Furthermore, chemical analysis by Fourier transform infrared spectroscopy revealed possible complexation of TA by Siaq, whereas UV-vis spectroscopy did not indicate an interaction of Siaq in the autoxidation process of TA. Investigation of additional metalloid ions showed that germanic acid was also able to initiate a continuous coating formation of TA, whereas boric acid prevented the polymerization process. In comparison to that of TA, the coating formation of pyrogallol (PG) and gallic acid (GA) was not affected by Siaq. PG formed continuous coatings also without Siaq, whereas GA formed only a monolayer in the presence of Siaq. However, Siaq induced a continuous layer formation of ellagic acid. These results indicate the specific importance of orthosilicic acid in the coating formation of polyphenolic molecules with multiple ortho-dihydroxy groups and open new possibilities to deposit TA on interfaces.

In Vitro Performance of Bioinspired Phenolic Nanocoatings for Endosseous Implant Applications.

In the quest for finding new strategies to enhance tissue integration and to reduce the risk of bacterial colonization around endosseous implants, we report the application of auto-oxidative phenolic coatings made of tannic acid and pyrogallol to titanium surfaces. The functionalized surfaces were screened for their biological performance using cultures of primary human osteoblasts and biofilm-forming bioluminescent staphylococci S. epidermidis Xen43 and S. aureus Xen29. No toxic effect of the coatings on osteoblasts was detected. While tannic acid coatings seemed to induce a delay in osteoblast maturation, they revealed anti-inflammatory potential. Similar effects were observed for pyrogallol coatings deposited for 24 h. Thin pyrogallol coatings deposited for 2 h seemed to promote osteoblast maturation and revealed increased calcium deposition. The effects on osteoblast were found to be related to the release of phenolic compounds from the surfaces. While the phenolic coatings could not inhibit staphylococcal biofilm formation on the titanium surfaces, released phenolic compounds had an inhibitory effect the growth of planktonic bacteria. In conclusion, the assessed coating systems represent a versatile functionalization method which exhibit promising effects for endosseous implant applications.

Real-time formation of salivary films onto polymeric materials for dental applications: Differences between unstimulated and stimulated saliva.

The formation of salivary films onto oral prostheses materials is of central importance for understanding their performance and interaction with oral tissue and flora. The aim of this work was to study and compare the salivary films formed from unstimulated and stimulated whole saliva on two common polymeric materials, polycarbonate and poly(methyl methacrylate). Irradiating these materials with UV light is a simple way to modify their wettability, roughness and ζ-potential. Therefore, the effect of UV exposure of polycarbonate and poly(methyl methacrylate) on saliva adsorption was also investigated. For this purpose a quartz crystal microbalance with dissipation and SDS-PAGE have been combined in order to associate the thicknesses and viscoelastic properties of the salivary films with their protein composition. SDS-PAGE results suggest that a larger diversity of proteins is involved in the formation of stimulated saliva pellicles. Furthermore, according to QCM-D, pellicles formed from stimulated saliva are thinner and stiffer than the ones formed from unstimulated saliva if the polymeric materials have not been exposed to UV light although both types of saliva form a biphasic layer. For UV-treated materials, the same is applied to polycarbonate but not to poly(methyl methacrylate) where stimulated saliva yields thicker and softer films than unstimulated saliva being the adsorption process of a multiphasic nature. These results highlight the importance of choosing the appropriate sample depending on the type of study to be performed.

Poly-l-lysine/heparin multilayer coatings prevent blood protein adsorption.

The adsorption of blood proteins, serum albumin (BSA), immunoglobulin G (IgG) and fibrinogen (FGN), onto model SiO2 planar surfaces coated with poly-l-lysine/heparin multilayers (PLL/HEP) has been investigated by means of ellipsometry and quartz crystal microbalance with dissipation. Aiming at the development of low fouling coatings, this study has been focused on the effects that the number of layers and the type of polyelectrolyte present on the topmost layer have on the adsorption of these proteins. The three proteins interact with PLL-ended coatings whereas HEP-ended coatings prevent the adsorption of both BSA and IgG and induce a decrease in the adsorbed amount of FGN, down to 0.4mg/m2 for three bilayers, as the number of PLL/HEP bilayers increases. These results suggest that heparin-ended multilayers prevent protein adsorption, which is an indicative of good blood compatibility. As a consequence we propose that PLL/HEP coatings could be used for the development of vascular medical devices.

Deposition Kinetics of Bioinspired Phenolic Coatings on Titanium Surfaces.

Polyphenols can form functional coatings on a variety of different materials through auto-oxidative surface polymerization in a manner similar to polydopamine coatings. However, the mechanisms behind the coating deposition are poorly understood. We report the coating deposition kinetics of the polyphenol tannic acid (TA) and the simple phenolic compound pyrogallol (PG) on titanium surfaces. The coating deposition was followed in real time over a period of 24 h using a quartz crystal microbalance with dissipation monitoring (QCM-D). TA coatings revealed a multiphasic layer formation: the deposition of an initial rigid layer was followed by the buildup of an increasingly dissipative layer, before mass adsorption stopped after approximately 5 h of coating time. The PG deposition was biphasic, starting with the adsorption of a nonrigid viscoelastic layer which was followed by layer stiffening upon further mass adsorption. Coating evaluation by ellipsometry and AFM confirmed the deposition kinetics determined by QCM-D and revealed maximum coating thicknesses of approximately 50 and 75 nm for TA and PG, respectively. Chemical characterization of the coatings and polymerized polyphenol particles indicated the involvement of both physical and chemical interactions in the auto-oxidation reactions.

Interfacial behavior and activity of laccase and bilirubin oxidase on bare gold surfaces.

Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL⁻¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm⁻², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O2) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s⁻¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s⁻¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes.

Tau protein provides DNA with thermodynamic and structural features which are similar to those found in histone-DNA complex.

Tau protein has been proposed as a trigger of Alzheimer's disease once it is hyperphosphorylated. However, the role that native tau forms play inside the neuronal nucleus remains unclear. In this work we present results concerning the interaction of tau protein with double-stranded DNA, single-stranded DNA, and also with a histone-DNA complex. The tau-DNA interaction results in a structure resembling the beads-on-a-string form produced by the binding of histone to DNA. DNA retardation assays show that tau and histone induce similar DNA retardation. A surface plasmon resonance study of tau-DNA interaction also confirms the minor groove of DNA as a binding site for tau, similarly to the histone binding. A residual binding of tau to DNA in the presence of Distamycin A, a minor groove binder, suggests the possibility that additional structural domains on DNA may be involved in tau interaction. Finally, DNA melting experiments show that, according to the Zipper model of helix-coil transition, the binding of tau increases the possibility of opening the DNA double helix in isolated points along the chain, upon increasing temperature. This behavior is analogous to histones and supports the previously reported idea that tau could play a protective role in stress situations. Taken together, these results show a similar behavior of tau and histone concerning DNA binding, suggesting that post-translational modifications on tau might impair the role that, by modulating the DNA function, might be attributable to the DNA-tau interaction.

Bioelectrocatalytic reduction of oxygen at gold nanoparticles modified with laccase.

To characterise bioelectrocatalytic oxygen reduction at gold nanoparticles (AuNPs) modified with Trametes hirsuta laccase (ThLc) combined electrochemical and quartz crystal microbalance measurements have been used. The electrodes with different degrees of AuNP-monolayer coverage, θ, have been studied. In every case of θ close to theoretically possible 44 ThLc molecules adsorbed at 22nm diameter AuNP. The bioelectrocatalytic current was recalculated down to the current at a single AuNP. Unexpectedly, the current at a single AuNP was higher when θ was higher. The maximum current reached at a single AuNP was 31·10(-18)A which corresponds to the enzyme turnover (kcat) 13s(-1). This rate is lower than the homogeneous ThLc turnover (190s(-1)) suggesting partial denaturation of ThLc upon adsorption or that some ThLc are not in DET contact with the electrode surface.

Specific binding of DNA to aggregated forms of Alzheimer's disease amyloid peptides.

Anomalous protein aggregation is closely associated to age-related mental illness. Extraneuronal plaques, mainly composed of aggregated amyloid peptides, are considered as hallmarks of Alzheimer's disease. According to the amyloid cascade hypothesis, this disease starts as a consequence of an abnormal processing of the amyloid precursor protein resulting in an excess of amyloid peptides. Nuclear localization of amyloid peptide aggregates together with amyloid-DNA interaction, have been repeatedly reported. In this paper we have used surface plasmon resonance and electron microscopy to study the structure and behavior of different peptides and proteins, including ß-lactoglobulin, bovine serum albumin, myoglobin, histone, casein and the amyloid-ß peptides related to Alzheimer's disease Aß25-35 and Aß1-40. The main purpose of this study is to investigate whether proneness to DNA interaction is a general property displayed by aggregated forms of proteins, or it is an interaction specifically related to the aggregated forms of those particular proteins and peptides related to neurodegenerative diseases. Our results reveal that those aggregates formed by amyloid peptides show a particular proneness to interact with DNA. They are the only aggregated structures capable of binding DNA, and show more affinity for DNA than for other polyanions like heparin and polyglutamic acid, therefore strengthening the hypothesis that amyloid peptides may, by means of interaction with nuclear DNA, contribute to the onset of Alzheimer's disease.

Influence of pH on the build-up of poly-L-lysine/heparin multilayers.

The effect of pH on the build-up of polyelectrolyte multilayers, PEMs, composed by poly-L-lysine and heparin onto two different substrates, silica and gold, has been studied by means of ellipsometry and quartz crystal microbalance with dissipation, QCM-D. Ellipsometry results indicate that the dry mass grows exponentially with the number of layers, and that this amount is larger as the pH values are raised. From QCM-D data the viscoelastic properties of the multilayered structure have been obtained. These data reflect that PEMs become more viscoelastic as the pH values are increased for silica substrates, while for gold the highest viscoelastic behavior is obtained at neutral pH and the elastic behavior becomes dominant as the pH is further increased or decreased. By combining these two surface techniques it has been also possible to determine the solvent content in the multilayers and reach a deeper understanding of the internal structure.

Alzheimer's disease amyloid peptides interact with DNA, as proved by surface plasmon resonance.

According to the amyloid hypothesis, abnormal processing of the ß-amyloid precursor protein in Alzheimer's disease patients increases the production of ß-amyloid toxic peptides, which, after forming highly aggregated fibrillar structures, lead to extracellular plaques formation, neuronal loss and dementia. However, a great deal of evidence has point to intracellular small oligomers of amyloid peptides, probably transient intermediates in the process of fibrillar structures formation, as the most toxic species. In order to study the amyloid-DNA interaction, we have selected here three different forms of the amyloid peptide: Aß1-40, Aß25-35 and a scrambled form of Aß25-35. Surface Plasmon Resonance was used together with UV-visible spectroscopy, Electrophoresis and Electronic Microscopy to carry out this study. Our results prove that, similarly to the full length Aß1-42, all conformations of toxic amyloid peptides, Aß1-40 and Aß25-35, may bind DNA. In contrast, the scrambled form of Aß25-35, a non-aggregating and nontoxic form of this peptide, could not bind DNA. We conclude that although the amyloid-DNA interaction is closely related to the amyloid aggregation proneness, this cannot be the only factor which determines the interaction, since small oligomers of amyloid peptides may also bind DNA if their predominant negatively charged amino acid residues are previously neutralized.

Friction force spectroscopy as a tool to study the strength and lateral diffusion of protein layers.

We present a method to study the strength of layers of biological molecules in liquid medium. The method is based on the Friction Force Spectroscopy operation mode of the Atomic Force Microscope. It works by scratching the sample surface at different applied loads while registering the evolution of the sample topography and of the friction between probe and sample. Results are presented for BSA and ß-casein monolayers on hydrophobic surfaces. We show how the simultaneous monitoring of topography and friction allows detecting differences not only between the strength of both types of layers, but also between the lateral diffusion of the proteins within these layers. Specifically, ß-casein is shown to form stronger layers than BSA. The yield strengths calculated for both of these systems are in the range 50-70 MPa. Moreover, while no lateral diffusion is observed for BSA, we show that ß-casein diffuses along the hydrophobic substrates at a rate higher than the scan velocity of the tip (16 µm s(-1) in our case).

Tau aggregation followed by atomic force microscopy and surface plasmon resonance, and single molecule tau-tau interaction probed by atomic force spectroscopy.

Intracellular neurofibrillary tangles, composed mainly of tau protein, and extracellular plaques, containing mostly amyloid-beta, are the two types of protein aggregates found upon autopsy within the brain of Alzheimer's disease patients. Polymers of tau protein can also be found in other neurodegenerative disorders known as tauopathies. Tau is a highly soluble protein, intrinsically devoid of secondary or tertiary structure, as many others proteins particularly prone to form fibrillar aggregations. The mechanism by which this unfolded molecule evolves to the well ordered helical filaments has been amply studied. In fact, it is a very slow process when followed in the absence of aggregation inducers. Herein we describe the use of surface plasmon resonance, atomic force microscopy, and atomic force spectroscopy to detect tau-tau interactions and to follow the process of aggregation in the absence of aggregation inducers. Tau-tau interactions are clearly detected, although a very long period of time is needed to observe filaments formation. Tau oligomers showing a granular appearance, however, are observed immediately as a consequence of this interaction. These granular tau oligomers slowly evolve to larger structures and eventually to filaments having a size smaller than those reported for paired helical filaments purified from Alzheimer's disease.

Interaction between Alzheimer's Abeta1-42 peptide and DNA detected by surface plasmon resonance.

Alzheimer's disease is a form of senile mental disorder characterized by the presence of extracellular plaques, containing amyloid-beta (Abeta) as the main component. According to the amyloid hypothesis, an increase of extracellular Abeta production is in the origin of the aberrant plaques causing neuronal loss and dementia. However, a wealth of evidence has been accumulated pointing to the toxicity of soluble intracellular Abeta, having different morphologies of aggregation, as the origin of the neurodegenerative process. The exact nature of the initial molecular events by which Abeta exerts its neurotoxicity, remains obscure. Different forms of soluble Abeta peptide aggregates have been recently found to reside in the nucleus of CHO cells and Alzheimer's disease brain samples. This paper focus mainly on the interaction between DNA and the 42 residue Abeta (Abeta42) as studied by Surface Plasmon Resonance. Electronic microscopy and UV-visible spectroscopy are also used to further characterize the interaction. Particular attention is paid to the extent of Abeta42 aggregation needed to observe the interaction with DNA. Our results show that DNA binds all soluble aggregate forms of Abeta42, therefore suggesting that DNA binding is a general property of different soluble forms of Abeta42, unrelated to the extent of aggregation.