Osteocytes Influence on Bone Matrix Integrity Affects Biomechanical Competence at Bone-Implant Interface of Bioactive-Coated Titanium Implants in Rat Tibiae.

Osseointegration is a prerequisite for the long-term success of implants. Titanium implants are preferred for their biocompatibility and mechanical properties. Nonetheless, the need for early and immediate loading requires enhancing these properties by adding bioactive coatings. In this preclinical study, extracellular matrix properties and cellular balance at the implant/bone interface was examined. Polyelectrolyte multilayers of chitosan and gelatin or with chitosan and Hyaluronic acid fabricated on titanium alloy using a layer-by-layer self-assembly process were compared with native titanium alloy. The study aimed to histologically evaluate bone parameters that correlate to the biomechanical anchorage enhancement resulted from bioactive coatings of titanium implants in a rat animal model. Superior collagen fiber arrangements and an increased number of active osteocytes reflected a significant improvement of bone matrix quality at the bone interface of the chitosan/gelatin-coated titan implants over chitosan/hyaluronic acid-coated and native implants. Furthermore, the numbers and localization of osteoblasts and osteoclasts in the reparative and remodeling phases suggested a better cellular balance in the chitosan/Gel-coated group over the other two groups. Investigating the micro-mechanical properties of bone tissue at the interface can elucidate detailed discrepancies between different promising bioactive coatings of titanium alloys to maximize their benefit in future medical applications.

How Nanotopography-Induced Conformational Changes of Fibrinogen Affect Platelet Adhesion and Activation.

The conformational state of adsorbed human plasma fibrinogen (HPF) has been recognized as the determinant factor in platelet adhesion and thrombus formation on blood-contacting biomaterials. Studies have highlighted the ability to control the HPF conformation merely by tailoring surface nanotopographical features. However, a clear relationship between the conformational changes of adsorbed HPF and the degree of platelet adhesion and activation achieved with different surface nanotopographies is still unclear. Here, we examined HPF assembly characteristics on nanostructured polybutene-1 (PB-1) surfaces with nanosized lamellar crystals (LCs), needle-like crystals (NLCs), and a nanostructured high-density polyethylene (HDPE) surface with shish-kebab crystals (SKCs), at a biologically relevant HPF concentration. By exposing the nanostructured surfaces with preadsorbed HPF to human platelets, significant differences in platelet response on LCs/SKCs and NLCs were identified. The former presented a uniform monolayer in the advanced stage of activation, whereas the latter exhibited minimal adhesion and the early stage of activation. Distinct platelet response was related to the postadsorption conformational changes in HPF, which were confirmed by topography-dependent shifts of the amide I band in attenuated total reflection-Fourier transform infrared (ATR-FTIR) analysis. Supported by atomic force microscopy (AFM) characterization, we propose that the mechanism behind the nanotopography-induced HPF conformation is driven by the interplay between the aspect ratios of polymeric crystals and HPF. From the biomedical perspective, our work reveals that surface structuring in a nanoscale size regime can provide a fine-tuning mechanism to manipulate HPF conformation, which can be exploited for the design of thromboresistant biomaterials surfaces.

Nanoconfinement and Sansetsukon-like Nanocrawling Govern Fibrinogen Dynamics and Self-Assembly on Nanostructured Polymeric Surfaces.

Surface nanostructures are increasingly more employed for controlled protein assembly on functional nanodevices, in nanobiotechnology, and in nanobiomaterials. However, the mechanism and dynamics of how nanostructures induce order in the adsorbed protein assemblies are still enigmatic. Here, we use single-molecule mapping by accumulated probe trajectories and complementary atomic force microscopy to shed light on the dynamic of in situ assembly of human plasma fibrinogen (HPF) adsorbed on nanostructured polybutene-1 (PB-1) and nanostructured polyethylene (PE) surfaces. We found a distinct lateral heterogeneity of HPF-polymer nanostructure interface (surface occupancy, residence time, and diffusion coefficient) that allow identifying the interplay between protein topographical nanoconfinement, protein diffusion mechanism, and ordered protein self-assembly. The protein diffusion analysis revealed high-diffusion polarization without correlation to the anisotropic friction characteristic of the polymer surfaces. This suggests that HPF molecules confined on the nanosized PB-1 needle crystals and PE shish-kebab crystals, respectively, undergo partial detachment and diffuse via a Sansetsukon-like nanocrawling mechanism. This mechanism is based on the intrinsic flexibility of HPF in the coiled-coil regions. We conclude that nanostructured surfaces that encourage this characteristic surface mobility are more likely to lead to the formation of ordered protein assemblies and may be useful for advanced nanobiomaterials.

Nanocrystal Width Controls Fibrinogen Orientation and Assembly Kinetics on Poly(butene-1) Surfaces.

From the view of biomedical relevance, it is known that a specific arrangement of surface-immobilized human plasma fibrinogen (HPF) molecules is required to retain their biological functionality. Here, we demonstrate a topographical effect of chemically identical isotactic poly(butene-1) (iPB-1) semicrystalline nanostructures on the adsorption behavior, i.e., conformation change and orientation of HPF molecules. Using the distinct crystallization of iPB-1 under different shear conditions, polymer thin films consisting of needle-like crystals (NLCs) or shish-kebab crystals (SKCs) having lateral dimension, i.e., width, smaller than or comparable to the HPF major axis, respectively, were fabricated. The protein adsorption kinetic studies by quartz crystal microbalance with dissipation (QCM-D) revealed surface-dependent packing density and assembly configuration of HPF. High-resolution imaging disclosed a "side-on" protein adsorption and anisotropic network formation on the NLCs. With a 2-fold orientation analysis performed at both "single" protein and multiprotein levels, we quantitatively proved the preferential alignment of adsorbed HPF molecules with respect to the axial direction of the NLCs. Remarkably, the iPB-1 surface with SKCs perturbed the "end-to-end" protein-protein interactions and thus hindered the network formation. The distinguished adsorption behavior of HPF molecules on iPB-1 surfaces is explained by the physical effect of crystal width, which is additionally supported by the synergistic effect of crystal curvature and aspect ratio. Our studies provide fundamental insight into purely topography-controlled self-assembly of HPF molecules, which might be further exploited in creating topographically defined implant surfaces for preventing protein aggregation related disorders.

pH-Dependent Ordered Fibrinogen Adsorption on Polyethylene Single Crystals.

Nanostructured surfaces have the potential to influence the assembly as well as the orientation of adsorbed proteins and may, thus, strongly influence the biomaterials' performance. For the class of polymeric (bio)materials a reproducible and well-characterized nanostructure is the ordered chain folded surface of a polyethylene single crystal (PE-SC). We tested the hypothesis that the trinodal-rod-shaped protein human plasma fibrinogen (HPF) adsorbs on the (001) surface of PE-SCs along specific crystallographic directions. The PE-SC samples were prepared by isothermal crystallization in dilute solution and characterized by atomic force microscopy (AFM) before as well as after HPF adsorption at different concentrations and pH values. At a physiological pH of 7.4, connected HPF molecules, or e.g., fibrils, fibril networks, or sponge-like structures, were observed on PE-SC surfaces that featured no preferential orientation. The observation of these nonoriented multiprotein assemblies was explained by predominant protein-protein interactions and limited surface diffusion. However, at an increased pH of 9.2, single HPF molecules, e.g., spherical-shaped and trinodal-rod-shaped HPF molecules as well as agglomerates, were observed on the PE-SC surface. The presence of single HPF molecules at increased pH was explained by decreased protein-protein interactions. These single trinodal-rod-shaped HPF molecules oriented preferentially along crystallographic [100] and [010] directions on the PE-SC surface which was explained by an increased amount of intermolecular bonds along these crystallographic directions with increased surface atom density. The study established that HPF molecules can align on chemically homogeneous surface topographies one order of magnitude smaller than the dimension of the protein. This advances the understanding of how to control the assembly and orientation of proteins on nanostructured polymer surfaces. Controlled protein adsorption is a crucial key to improve the surface functionality of future implants and biosensors.

The antimicrobial effect of calcium-doped titanium is activated by fibrinogen adsorption.

Directly targeting bacterial cells is the present paradigm for designing antimicrobial biomaterial surfaces and minimizing device-associated infections (DAIs); however, such pathways may create problems in tissue integration because materials that are toxic to bacteria can also be harmful to mammalian cells. Herein, we report an unexpected antimicrobial effect of calcium-doped titanium, which itself has no apparent killing effect on the growth of pathogenic bacteria (Pseudomonas aeruginosa, Pa, ATCC 27853) while presenting strong inhibition efficiency on bacterial colonization after fibrinogen adsorption onto the material. Fine X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy analyses reported calcium-dependent shifts of the binding energy in nitrogen and oxygen involved groups and wavenumbers in the amide I and II bands of the adsorbent fibrinogen, demonstrating that locally delivered calcium can react with the carboxy-terminal regions of the Aα chains and influence their interaction with the N-termini of the Bß chains in fibrinogen. These reactions facilitate the exposure of the antimicrobial motifs of the protein, indicating the reason for the surprising antimicrobial efficacy of calcium-doped titanium. Since protein adsorption is an immediate intrinsic step during the implantation surgery, this finding may shift the present paradigm on the design of implantable antibacterial biomaterial surfaces.

Self-assembled fibrinogen-fibronectin hybrid protein nanofibers with medium-sensitive stability.

Hybrid protein nanofibers (hPNFs) have been identified as promising nano building blocks for numerous applications in nanomedicine and tissue engineering. We have recently reported a nature-inspired, self-assembly route to create hPNFs from human plasma proteins, i.e., albumin and hemoglobin. However, it is still unclear whether the same route can be applied to other plasma proteins and whether it is possible to control the composition of the resulting fibers. In this context, to further understand the hPNFs self-assembly mechanism and to optimize their properties, we report herein on ethanol-induced self-assembly of two different plasma proteins, i.e., fibrinogen (FG) and fibronectin (FN). We show that by varying initial protein ratios, the composition and thus the properties of the resulting hPNFs can be fine-tuned. Specifically, atomic force microscopy, hydrodynamic diameter, and zeta potential data together revealed a strong correlation of the hPNFs dimensions and surface charge to their initial protein mixing ratio. The composition-independent prompt dissolution of hPNFs in ultrapure water, in contrast to their stability in PBS, indicates that the molecular arrangement of FN and FG in hPNFs is mainly based on electrostatic interactions. Supported by experimental data we introduce a feasible mechanism that explains the interactions between FN and FG and their self-assembly to hPNFs. These findings contribute to the understanding of dual protein interactions, which can be beneficial in designing innovative biomaterials with multifaceted biological and physical characteristics.

Quantifying the relationship between surfaces' nano-contact point density and adhesion force of Candida albicans.

It has been recently recognized that controlled surface structuring on the nanometer scale is a successful strategy to endow different materials with antimicrobial properties. Despite many studies on bacterial interactions with nanostructured surfaces, a quantitative link between surface topography and bacterial adhesion is still missing. To quantitatively link cell adhesion data with topographical surface parameters, we performed single-cell spectroscopy on chemically identical surfaces with controlled nano-contact point density achieved by immobilization of gold nanoparticles (AuNP) on gold thin films. Such materials surfaces have previously shown antimicrobial (anti-adhesive) efficacy towards Gram-negative Escherichia coli cells. In the current study, the influence of nano-structured surfaces on the surface coverage and adhesion forces of clinically relevant Candida albicans (C. albicans), the fungus primarily associated with implant infections, was investigated to validate their antimicrobial potency against different microbial cells. The adhesion forces of C. albicans cells to nanostructured surfaces showed a decreasing trend with decreasing contact-point density and correlated well with the results of the respective C. albicans cell counts. The surfaces with the lowest contact-point density, 25 AuNP/µm², resulted in an average adhesion force of 5 nN, which was up to 5 times lower compared to control and 61 AuNP/µm² surfaces. Further, detailed analyses of force-distance curves revealed that the work of adhesion, and thus the energy required to remove the C. albicans cell from the surface is up to 10 times lower on 25 AuNP/µm² surfaces compared to unstructured surfaces. These findings show that a controlled tuning of nanostructured surfaces in terms of accessible nano-contact points is crucial to generate surface structures with enhanced antimicrobial properties. The gained knowledge can be further exploited for the design of biomaterials surfaces to prevent adhesion of some most commonly encountered pathogens.

Effect of an electric field during the deposition of silicon dioxide thin films by plasma enhanced atomic layer deposition: an experimental and computational study.

The growth, chemical, structural, mechanical, and optical properties of oxide thin films deposited by plasma enhanced atomic layer deposition (PEALD) are strongly influenced by the average-bias voltage applied during the reaction step of surface functional groups with oxygen plasma species. Here, this effect is investigated thoroughly for SiO2 deposited in two different PEALD tools at average-bias voltages up to -300 V. Already at a very low average-bias voltage (< -10 V), the SiO2 films have significantly lower water content than films grown without biasing together with the formation of denser films having a higher refractive index and nearly stoichiometric composition. Substrate biasing during PEALD also enables control of mechanical stress. The experimental findings are supported by density functional theory and atomistic simulations. They demonstrate that the application of an electric field during the plasma step results in an increased energy transfer between energetic ions and the surface, directly influencing relevant surface reactions. Applying an electric field during the PEALD process leads to SiO2 thin films with significantly improved properties comparable to films grown by ion beam sputtering.

Protein Handshake on the Nanoscale: How Albumin and Hemoglobin Self-Assemble into Nanohybrid Fibers.

Creating and establishing proof of hybrid protein nanofibers (hPNFs), i.e., PNFs that contain more than one protein, is a currently unsolved challenge in bioinspired materials science. Such hPNFs could serve as universal building blocks for the bottom-up preparation of functional materials with bespoke properties. Here, inspired by the protein assemblies occurring in nature, we introduce hPNFs created via a facile self-assembly route and composed of human serum albumin (HSA) and human hemoglobin (HGB) proteins. Our circular dichroism results shed light on the mechanism of the proteins' self-assembly into hybrid nanofibers, which is driven by electrostatic/hydrophobic interactions between similar amino acid sequences (protein handshake) exposed to ethanol-triggered protein denaturation. Based on nanoscale characterization with tip-enhanced Raman spectroscopy (TERS) and immunogold labeling, our results demonstrate the existence and heterogenic nature of the hPNFs and reveal the high HSA/HGB composition ratio, which is attributed to the fast self-assembling kinetics of HSA. The self-assembled hPNFs with a high aspect ratio of over 100 can potentially serve as biocompatible units to create larger bioactive structures, devices, and sensors.