Surface Chemistry Induced IgG Unfolding and Modulation of Immune Responses.

Immunoglobulin G (IgG) comprises a significant portion of the protein corona that forms on biomaterial surfaces and holds a pivotal role in modulating host immune responses. To shed light on the important relationship between biomaterial surface functionality, IgG adsorption, and innate immune responses, we prepared, using plasma deposition, four surface coatings with specific chemistries, wettability, and charge. We found that nitrogen-containing coatings such as these deposited from allylamine (AM) and 2-methyl-2-oxazoline (POX) cause the greatest IgG unfolding, while hydrophilic acrylic acid (AC) surfaces allowed for the retention of the protein structure. Structural changes in IgG significantly modulated macrophage attachment, migration, polarization, and the expression of pro- and anti-inflammatory cytokines. Unfolded IgG on the POX and AM surfaces enhanced macrophage attachment, migration, extracellular trap release, and pro-inflammatory factors production such as IL-6 and TNF-α. Retention of IgG structure on the AC surface downregulated inflammatory responses. The findings of this study demonstrate that the retention of protein structure is an essential factor that must be taken into consideration when designing biomaterial surfaces. Our study indicates that using hydrophilic surface coatings could be a promising strategy for designing immune-modulatory biomaterials for clinical applications.

Exploring the Challenges of Characterising Surface Topography of Polymer-Nanoparticle Composites.

Nanomechanical testing plays a crucial role in evaluating surfaces containing nanoparticles. Testing verifies surface performance concerning their intended function and detects any potential shortcomings in operational standards. Recognising that nanostructured surfaces are not always straightforward or uniform is essential. The chemical composition and morphology of these surfaces determine the end-point functionality. This can entail a layered surface using materials in contrast to each other that may require further modification after nanomechanical testing to pass performance and quality standards. Nanomechanical analysis of a structured surface consisting of a poly-methyl oxazoline film base functionalised with colloidal gold nanoparticles was demonstrated using an atomic force microscope (AFM). AFM nanomechanical testing investigated the overall substrate architecture's topographical, friction, adhesion, and wear parameters. Limitations towards its potential operation as a biomaterial were also addressed. This was demonstrated by using the AFM cantilever to apply various forces and break the bonds between the polymer film and gold nanoparticles. The AFM instrument offers an insight to the behaviour of low-modulus surface against a higher-modulus nanoparticle. This paper details the bonding and reaction limitations between these materials on the application of an externally applied force. The application of this interaction is highly scrutinised to highlight the potential limitations of a functionalised surface. These findings highlight the importance of conducting comprehensive nanomechanical testing to address concerns related to fabricating intricate biomaterial surfaces featuring nanostructures.

Damage Behavior with Atomic Force Microscopy on Anti-Bacterial Nanostructure Arrays.

The atomic force microscope is a versatile tool for assessing the topography, friction, and roughness of a broad spectrum of surfaces, encompassing anti-bacterial nanostructure arrays. Measuring and comparing all these values with one instrument allows clear comparisons of many nanomechanical reactions and anomalies. Increasing nano-Newton-level forces through the cantilever tip allows for the testing and measuring of failure points, damage behavior, and functionality under unfavorable conditions. Subjecting a grade 5 titanium alloy to hydrothermally etched nanostructures while applying elevated cantilever tip forces resulted in the observation of irreversible damage through atomic force microscopy. Despite the damage, a rough and non-uniform morphology remained that may still allow it to perform in its intended application as an anti-bacterial implant surface. Utilizing an atomic force microscope enables the evaluation of these surfaces before their biomedical application.

Vancomycin tolerance of adherent Staphylococcus aureus is impeded by nanospike-induced physiological changes.

Bacterial colonization of implantable biomaterials is an ever-pervasive threat that causes devastating infections, yet continues to elude resolution. In the present study, we report how a rationally designed antibacterial surface containing sharp nanospikes can enhance the susceptibility of pathogenic bacteria to antibiotics used in prophylactic procedures. We show that Staphylococcus aureus, once adhered to a titanium surface, changes its cell-surface charge to increase its tolerance to vancomycin. However, if the Ti surface is modified to bear sharp nanospikes, the activity of vancomycin is rejuvenated, leading to increased bacterial cell death through synergistic activity. Analysis of differential gene expression provided evidence of a set of genes involved with the modification of cell surface charge. Synchrotron-sourced attenuated Fourier-transform infrared microspectroscopy (ATR-FTIR), together with multivariate analysis, was utilized to further elucidate the biochemical changes of S. aureus adhered to nanospikes. By inhibiting the ability of the pathogen to reduce its net negative charge, the nanoengineered surface renders S. aureus more susceptible to positively charged antimicrobials such as vancomycin. This finding highlights the opportunity to enhance the potency of prophylactic antibiotic treatments during implant placement surgery by employing devices having surfaces modified with spike-like nanostructures.

Interplay between Immune and Bacterial Cells on a Biomimetic Nanostructured Surface: A "Race for the Surface" Study.

Biomaterial-associated infection is an ever-increasing risk with devasting consequences for patients. Considerable research has been undertaken to address this issue by imparting antibacterial properties to the surface of biomedical implants. One approach that generated much interest over recent years was the generation of bioinspired bactericidal nanostructures. In the present report, we have investigated the interplay between macrophages and bacteria on antibacterial nanostructured surfaces to determine the outcome of the so-called "race for the surface". Our results showed that macrophages can indeed outcompete Staphylococcus aureus via multiple mechanisms. The early generation of reactive oxygen species by macrophages, downregulation of bacterial virulence gene expression, and the bactericidal nature of the nanostructured surface itself collectively acted to help the macrophage to win the race. This study highlights the potential of nanostructured surfaces to reduce infection rates and improve the long-term success of biomedical implants. This work can also serve as guidance to others to investigate in vitro host-bacteria interactions on other candidate antibacterial surfaces.

Printable Hydrogel Arrays for Portable and High-Throughput Shear-Mediated Assays.

Hydrogels have been widely used to entrap biomolecules for various biocatalytic reactions. However, solute diffusion in these matrices to initiate such reactions can be a very slow process. Conventional mixing remains a challenge as it can cause irreversible distortion or fragmentation of the hydrogel itself. To overcome the diffusion-limit, a shear-stress-mediated platform named the portable vortex-fluidic device (P-VFD) is developed. P-VFD is a portable platform which consists of two main components, (i) a plasma oxazoline-coated polyvinyl chloride (POx-PVC) film with polyacrylamide and alginate (PAAm/Alg-Ca2+) tough hydrogel covalently bound to its surface and (ii) a reactor tube (L × D: 90 mm × 20 mm) where the aforementioned POx-PVC film could be readily inserted for reactions. Through a spotting machine, the PAAm/Alg-Ca2+ hydrogel can be readily printed on a POx-PVC film in an array pattern and up to 25.4 J/m2 adhesion energy can be achieved. The hydrogel arrays on the film not only offer a strong matrix for entrapping biomolecules such as streptavidin-horseradish peroxidase but are also shear stress-tolerant in the reactor tube, enabling a >6-fold increase in its reaction rate after adding tetramethylbenzidine, relative to incubation. Through using the tough hydrogel and its stably bonded substrate, this portable platform effectively overcomes the diffusion-limit and achieves fast assay detection without causing appreciable hydrogel array deformation or dislocation on the substrate film.

Antibacterial Nanostructured Surfaces Modulate Protein Adsorption, Inflammatory Responses, and Fibrous Capsule Formation.

The present study interrogates the interaction of highly efficient antibacterial surfaces containing sharp nanostructures with blood proteins and the subsequent immunological consequences, processes that are of key importance for the fate of every implantable biomaterial. Studies with human serum and plasma pointed to significant differences in the composition of the protein corona that formed on control and nanostructured surfaces. Quantitative analysis using liquid chromatography-mass spectrometry demonstrated that the nanostructured surface attracted more vitronectin and less complement proteins compared to the untreated control. In turn, the protein corona composition modulated the adhesion and cytokine expression by immune cells. Monocytes produced lower amounts of pro-inflammatory cytokines and expressed more anti-inflammatory factors on the nanostructured surface. Studies using an in vivo subcutaneous mouse model showed reduced fibrous capsule thickness which could be a consequence of the attenuated inflammatory response. The results from this work suggest that antibacterial surface modification with sharp spike-like nanostructures may not only lead to the reduction of inflammation but also more favorable foreign body response and enhanced healing, processes that are beneficial for most medical devices implanted in patients.

Preventing Peri-implantitis: The Quest for a Next Generation of Titanium Dental Implants.

Titanium and its alloys are frequently the biomaterial of choice for dental implant applications. Although titanium dental implants have been utilized for decades, there are yet unresolved issues pertaining to implant failure. Dental implant failure can arise either through wear and fatigue of the implant itself or peri-implant disease and subsequent host inflammation. In the present report, we provide a comprehensive review of titanium and its alloys in the context of dental implant material, and how surface properties influence the rate of bacterial colonization and peri-implant disease. Details are provided on the various periodontal pathogens implicated in peri-implantitis, their adhesive behavior, and how this relationship is governed by the implant surface properties. Issues of osteointegration and immunomodulation are also discussed in relation to titanium dental implants. Some impediments in the commercial translation for a novel titanium-based dental implant from "bench to bedside" are discussed. Numerous in vitro studies on novel materials, processing techniques, and methodologies performed on dental implants have been highlighted. The present report review that comprehensively compares the in vitro, in vivo, and clinical studies of titanium and its alloys for dental implants.

Surfaces Containing Sharp Nanostructures Enhance Antibiotic Efficacy.

The ever-increasing rate of medical device implantations is met by a proportionately high burden of implant-associated infections. To mitigate this threat, much research has been directed toward the development of antibacterial surface modifications by various means. One recent approach involves surfaces containing sharp nanostructures capable of killing bacteria upon contact. Herein, we report that the mechanical interaction between Staphylococcus aureus and such surface nanostructures leads to a sensitization of the pathogen to the glycopeptide antibiotic vancomycin. We demonstrate that this is due to cell wall damage and impeded bacterial defenses against reactive oxygen species. The results of this study promise to be impactful in the clinic, as a combination of nanostructured antibacterial surfaces and antibiotics commonly used in hospitals may improve antimicrobial therapy strategies, helping clinicians to prevent and treat implant-associated infections using reduced antibiotic concentrations instead of relying on invasive revision surgeries with often poor outcomes.

Nanomechanical tribological characterisation of nanostructured titanium alloy surfaces using AFM: A friction vs velocity study.

Medical-grade titanium alloys used for orthopaedic implants are at risk from infections and complications such as wear and tear. We have recently shown that hydrothermally etched (HTE) nanostructures (NS) formed on the Ti6AlV4 alloy surfaces impart enhanced anti-bacterial activity which results in inhibited formation of bacterial biofilm. Although these titanium alloy nanostructures may resist bacterial colonisation, their frictional properties are yet to be understood. Orthopaedic devices are encapsulated by bone and muscle tissue. Contact friction between orthopaedic implant surfaces and these host tissues may trigger inflammation, osteolysis and wear. To address these challenges, we performed simulation of the contact behaviour between a smooth control Ti6Al4V alloy and HTE surfaces against a hardwearing SiO2 sphere using Atomic Force Microscopy (AFM) in Lateral Force Microscopy mode. The friction study was evaluated in both air and liquid environments at high (5 Hz) and low (0.5 Hz) scan velocities. Lower scan velocities demonstrated opposing friction force changes between the two mediums, with friction stabilising at higher velocities. The friction measured on the NS alloy surfaces was reduced by ~20% in air and ~80% in phosphate buffered saline, in comparison to the smooth control surface, displaying a non-linear behaviour of the force applied by the AFM tip. Changes in friction values and cantilever scan velocities on different substrates are discussed with respect to the Stribeck curve. Reduced friction on nanostructured surfaces may improve wear resistance and aid osseointegration.

Bio-Inspired Nanostructured Ti-6Al-4V Alloy: The Role of Two Alkaline Etchants and the Hydrothermal Processing Duration on Antibacterial Activity.

Inspired by observations that the natural topography observed on cicada and dragonfly wings may be lethal to bacteria, researchers have sought to reproduce these nanostructures on biomaterials with the goal of reducing implant-associated infections. Titanium and its alloys are widely employed biomaterials with excellent properties but are susceptible to bacterial colonisation. Hydrothermal etching is a simple, cost-effective procedure which fabricates nanoscale protrusions of various dimensions upon titanium, depending on the etching parameters used. We investigated the role of etching time and the choice of cation (sodium and potassium) in the alkaline heat treatment on the topographical, physical, and bactericidal properties of the resulting modified titanium surfaces. Optimal etching times were 4 h for sodium hydroxide (NaOH) and 5 h for potassium hydroxide (KOH). NaOH etching for 4 h produced dense, but somewhat ordered, surface nanofeatures with 75 nanospikes per µm2. In comparison, KOH etching for 5 h resulted sparser but nonetheless disordered surface morphology with only 8 spikes per µm2. The NaOH surface was more effective at eliminating Gram-negative pathogens, while the KOH surface was more effective against the Gram-positive strains. These findings may guide further research and development of bactericidal titanium surfaces which are optimised for the predominant pathogens associated with the intended application.

Long-term antibacterial properties of a nanostructured titanium alloy surface: An in vitro study.

The demand for joint replacement and other orthopedic surgeries involving titanium implants is continuously increasing; however, 1%-2% of surgeries result in costly and devastating implant associated infections (IAIs). Pseudomonas aeruginosa and Staphylococcus aureus are two common pathogens known to colonise implants, leading to serious complications. Bioinspired surfaces with spike-like nanotopography have previously been shown to kill bacteria upon contact; however, the longer-term potential of such surfaces to prevent or delay biofilm formation is unclear. Hence, we monitored biofilm formation on control and nanostructured titanium disc surfaces over 21 days following inoculation with Pseudomonas aeruginosa and Staphylococcus aureus. We found a consistent 2-log or higher reduction in live bacteria throughout the time course for both bacteria. The biovolume on nanostructured discs was also significantly lower than control discs at all time points for both bacteria. Analysis of the biovolume revealed that for the nanostructured surface, bacteria was killed not just on the surface, but at locations above the surface. Interestingly, pockets of bacterial regrowth on top of the biomass occurred in both bacterial species, however this was more pronounced for S. aureus cultures after 21 days. We found that the nanostructured surface showed antibacterial properties throughout this longitudinal study. To our knowledge this is the first in vitro study to show reduction in the viability of bacterial colonisation on a nanostructured surface over a clinically relevant time frame, providing potential to reduce the likelihood of implant associated infections.

In Vitro Bactericidal Efficacy of Nanostructured Ti6Al4V Surfaces is Bacterial Load Dependent.

The demand for medical implants globally has increased significantly due to an aging population amongst other reasons. Despite the overall increase in the survivorship of Ti6Al4V implants, implant infection rates are increasing due to factors such as diabetes, obesity, and bacterial resistance to antibiotics. Two commonly found bacteria implicated in implant infections are Staphylococcus aureus and Pseudomonas aeruginosa. Based on prior work that showed nanostructured surfaces might have potential in passively killing these bacterial species, we developed a hierarchical, hydrothermally etched, nanostructured titanium surface. To evaluate the antibacterial efficacy of this surface, etched and as-received surfaces were inoculated with S. aureus or P. aeruginosa at concentrations ranging from 102 to 109 colony-forming units per disc. Live/dead staining revealed there was a 60% decrease in viability for S. aureus and greater than a 98% decrease for P. aeruginosa on etched surfaces at the lowest inoculum of 102 CFU/disc, when compared to the control surface. Bactericidal efficiency decreased with increasing bacterial concentrations in a stepwise manner, with decreases in bacterial viability noted for S. aureus above 105 CFU/disc and above 106 CFU/disc for P. aeruginosa. Surprisingly, biofilm depth analysis revealed a decrease in bacterial viability in the 2 µm layer furthest from the nanostructured surface. The nanostructured Ti6Al4V surface developed here holds the potential to reduce the rate of implant infections.

Herceptin functionalized microfluidic polydimethylsiloxane devices for the capture of human epidermal growth factor receptor 2 positive circulating breast cancer cells.

Building on recent breakthroughs in the field of microfluidic-based capture of rare cancer cells circulating in the blood, the present article reports on the use of Herceptin functionalized PDMS devices designed to efficiently capture from blood cancer cells, overexpressing the tyrosine kinase human epidermal growth factor receptor (HER2). The identification of patients overexpressing HER2 is critical as it typically associates with an aggressive disease course in breast cancer and poor prognosis. Importantly, HER2 positive patients have been found to significantly benefit from Herceptin (Trastuzumab), a humanized monoclonal antibody (MAb) against HER2. Disposable PDMS devices prepared using standard soft lithography were functionalized by the plasma polymerization of an epoxy-containing monomer. The epoxy-rich thin film (AGEpp) thus created could be conjugated with Herceptin either directly or through a polyethylene glycol interlayer. The properties and reactivity toward the monoclonal antibody conjugation of these coatings were determined using x-ray photoelectron spectroscopy; direct conjugation provided a good compromise in reactivity and resistance to biologically nonspecific fouling and was selected. Using the breast cancer cell line SK-BR-3 as a model for cells overexpressing HER2, the immunocapture efficacy of the Herceptin functionalized PDMS was demonstrated in model studies. Validation studies confirmed the ability of the device to efficiently capture (∼80% capture yield) HER2 positive cells from full blood.

Electrochemically induced growth of zinc oxide.

The electrolytically induced precipitation of zinc oxide from zinc nitrate solution on gold surfaces in the presence of water-soluble polymers was examined for reaction times between 0.5 and 600 seconds. Regardless of the additive, polycrystalline films of zinc oxide have formed after 30 seconds, but polymeric additives dramatically change the morphology of the ZnO films. Amperometric analysis and fitting the diffusion reduced the current density-time curve according to Avrami kinetics and it reveals that polymers bearing methacrylic acid groups result in spherical growth whereas such with sulfonic acid groups lead to a platelike growth of crystallites. Without additive prisms grow predominantly in one dimension. These findings are confirmed also by scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis.

High-resolution in situ x-ray study of the hydrophobic gap at the water-octadecyl-trichlorosilane interface.

The knowledge of the microscopic structure of water at interfaces is essential for the understanding of interfacial phenomena in numerous natural and technological environments. To study deeply buried liquid water-solid interfaces, high-energy x-ray reflectivity measurements have been performed. Silicon wafers, functionalized by a self-assembled monolayer of octadecyl-trichlorosilane, provide strongly hydrophobic substrates. We show interfacial density profiles with angstrom resolution near the solid-liquid interface of water in contact with an octadecyl-trichlorosilane layer. The experimental data provide clear evidence for the existence of a hydrophobic gap on the molecular scale with an integrated density deficit rhod = 1.1 A g cm(-3) at the solid-water interface. In addition, measurements on the influence of gases (Ar, Xe, Kr, N(2), O(2), CO, and CO(2)) and HCl, dissolved in the water, have been performed. No effect on the hydrophobic water gap was found.

Directed crystallisation of zinc oxide on patterned surfaces.

Patterned self-assembled monolayers of functionalised alkane thiols were prepared on gold substrates, using UV-photolithography. Two alkane thiols, 11-mercaptoundecanoic acid (MUA) and a fluorinated decane thiol (FDT, CF3(CF2)7CH2CH2SH) were used to fabricate chemically structured surfaces which served as templates for zinc oxide (ZnO) crystallisation. When these patterns, containing high (MUA) and low (FDT) surface energy regions were exposed to a 10 mM zinc nitrate crystallising solution, nucleation occurred selectively on the low energy regions. After 90 min, hexagonal prisms had grown upright on these areas. The crystal growth is uniform with a crystal length of about 1 mum and a diameter between 50 and 100 nm. We attribute the selective growth to a combination of crystallographic frustration of the zinc ions on the high energy regions and an accumulation of hydroxide ions on the low energy regions.