Sidónio C. Freitas

Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation.

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin-thrombin complex to minimize the biotinylation of the residues involved in thrombin-boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using ¹²⁵I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilins potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.

Adhesion of human leukocytes on mixtures of hydroxyl‐ and methyl‐terminated self‐assembled monolayers: Effect of blood protein adsorption

The adhesion of human leukocytes to nanostructured surfaces with different chemical properties and the effect of protein adsorption were investigated. Self-assembled monolayers (SAMs) prepared with mixtures of methyl- and hydroxyl-terminated alkanethiols in different percentages on gold were used. The surfaces were pre-immersed in distinct protein solutions (human serum albumin, human fibrinogen, and autologous plasma). Adherent leukocytes were analyzed both by light and SEM. SAMs submitted to pre-immersion in plasma presented higher numbers of adherent leukocytes in the pure OH-terminated SAM, whereas methyl-terminated surfaces accounted for the lowest number of adherent cells. We observed a general increase in the number of adherent human leukocytes as the percentage of OH groups on the surface of the SAMs increased for all the pre-immersion conditions investigated. The number of adherent human leukocytes is highly influenced by the pre-immersion conditions used, and this observation is particularly relevant in the case of the methyl-terminated SAMs. The results obtained demonstrate that surface chemistry has a major influence in leukocyte adhesion to biomaterials, and that pre-immersion in protein solutions has a determinant effect in leukocyte adhesion.

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices.

Silica Sol-Gel Patterned Surfaces Based on Dip-Pen Nanolithography and Microstamping: A Comparison in Resolution and Throughput

Fabrication of patterns on silicon and gold via Dip-Pen Nanolithography (DPN) using silica sol as ink and the combination of DPN, soft lithography, and silica sol-gel to transfer patterns from silicon and gold to stainless steel were assessed. In addition, a comparison in terms of throughput and resolution of both protocols was performed. Optical, scanning electron and atomic force microscopy were used to characterize the patterns. Silica sol showed high resolution but low throughput when used to pattern directly on gold and silicon using DPN. The combination of DPN, silica sol-gel and soft lithography showed high throughput and resolution. The present experimental methodology was useful to create patterns on a surface and transfer them to another surface of interest, which may serve as a biomaterial surface modification model.

A novel approach to create an antibacterial surface using titanium dioxide and a combination of dip-pen nanolithography and soft lithography

Soft lithography and Dip-Pen Nanolithography (DPN) are techniques that have been used to modify the surface of biomaterials. Modified surfaces play a role in reducing bacterial adhesion and biofilm formation. Also, titanium dioxide has been reported as an antibacterial substance due to its photocatalytic effect. This work aimed at creating patterns on model surfaces using DPN and soft lithography combined with titanium dioxide to create functional antibacterial micropatterned surfaces, which were tested against Streptococcus mutans. DPN was used to create a master pattern onto a model surface and microstamping was performed to duplicate and transfer such patterns to medical-grade stainless steel 316L using a suspension of TiO2. Modified SS316L plates were subjected to UVA black light as photocatalytic activator. Patterns were characterized by atomic force microscopy and biologically evaluated using S. mutans. A significant reduction of up to 60% in bacterial adhesion to TiO2 -coated and -micropatterned surfaces was observed. Moreover, both TiO2 surfaces reduced the viability of adhered bacteria after UV exposure. TiO2 micropatterned demonstrated a synergic effect between physical and chemical modification against S. mutans. This dual effect was enhanced by increasing TiO2 concentration. This novel approach may be a promising alternative to reduce bacterial adhesion to surfaces.

Conjugation Chemistry Principles and Surface Functionalization of Nanomaterials

Abstract The (bio)conjugation principles that can be applied to functionalize (nano)materials in the context of biomedical applications are vast. In fact, the (bio)conjugation field has particularly advanced at an astonishing pace in the last decades. Instead of being exhaustive, here we aimed to present the basic toolbox that is currently available to a researcher to move in the (bio)functionalization area, particularly focusing on common requisites of the biomedical applications. Some of these tools are further discussed in the context of the surface (bio)functionalization of self-assembled monolayers because these are frequently used as proof-of-concept model surfaces for detailed studies, prior extrapolation to more complex ones. Finally, we discuss the challenges that lie ahead in the (bio)conjugation field toward achieving more efficient syntheses, the simplification of purification steps and the improvement of the biocompatibility of the final products.

Soft Lithography and Minimally Human Invasive Technique for Rapid Screening of Oral Biofilm Formation on New Microfabricated Dental Material Surfaces

Introduction Microfabrication offers opportunities to study surface concepts focused to reduce bacterial adhesion on implants using human minimally invasive rapid screening (hMIRS). Wide information is available about cell/biomaterial interactions using eukaryotic and prokaryotic cells on surfaces of dental materials with different topographies, but studies using human being are still limited. Objective To evaluate a synergy of microfabrication and hMIRS to study the bacterial adhesion on micropatterned surfaces for dental materials. Materials and Methods Micropatterned and flat surfaces on biomedical PDMS disks were produced by soft lithography. The hMIRS approach was used to evaluate the total oral bacterial adhesion on PDMS surfaces placed in the oral cavity of five volunteers (the study was approved by the University Ethical Committee). After 24 h, the disks were analyzed using MTT assay and light microscopy. Results In the present pilot study, microwell structures were microfabricated on the PDMS surface via soft lithography with a spacing of 5 µm. Overall, bacterial adhesion did not significantly differ between the flat and micropatterned surfaces. However, individual analysis of two subjects showed greater bacterial adhesion on the micropatterned surfaces than on the flat surfaces. Significance Microfabrication and hMIRS might be implemented to study the cell/biomaterial interactions for dental materials.

Self-Assembled Monolayers for Dental Implants

Implant-based therapy is a mature approach to recover the health conditions of patients affected by edentulism. Thousands of dental implants are placed each year since their introduction in the 80s. However, implantology faces challenges that require more research strategies such as new support therapies for a world population with a continuous increase of life expectancy, to control periodontal status and new bioactive surfaces for implants. The present review is focused on self-assembled monolayers (SAMs) for dental implant materials as a nanoscale-processing approach to modify titanium surfaces. SAMs represent an easy, accurate, and precise approach to modify surface properties. These are stable, well-defined, and well-organized organic structures that allow to control the chemical properties of the interface at the molecular scale. The ability to control the composition and properties of SAMs precisely through synthesis (i.e., the synthetic chemistry of organic compounds with a wide range of functional groups is well established and in general very simple, being commercially available), combined with the simple methods to pattern their functional groups on complex geometry appliances, makes them a good system for fundamental studies regarding the interaction between surfaces, proteins, and cells, as well as to engineering surfaces in order to develop new biomaterials.

Solvothermal Synthesis of Magnesium Oxide-Substituted Hydroxyapatite Nanoparticles as Antibacterial Nanomaterial for Biomedical Applications

In order to generate bactericidal effects in the oral cavity, several alternatives have been studied, including the use of silver nanoparticles but presents problems such as toxicity and low biocompatibility. From human-inspired systems, the antibacterial efficiency of the hydroxyapatite nanoparticles depends strongly on the type of composites and nanoparticles size. Several types of hydroxyapatite nanoparticles and their derivatives have received much attention for their antibacterial potential effect, including magnesium oxide nanoparticles. The purpose of this research was to produce a biocompatible antimicrobial compound of nanoparticles of hydroxyapatite doped with magnesium oxide to generate antibacterial effects in the oral cavity. The solvothermal method was used to produce hydroxyapatite nanoparticles doped with magnesium oxide. Antibacterial activity of as synthesized nanopowders against cariogenic Streptococcus mutans was tested by the CLSI disk-diffusion method. As result of this research, hydroxyapatite doped with magnesium nanoparticles (nHAMg) were successfully synthetized by the solvothermal method where in structural characterization indicates magnesium substitution and FTIR analysis gives a broader spectrum of the nHAMg when compared to pure nHA and crystallite size of nHA decreased. Furthermore, results of antibacterial assays showed that nHAMg allow to inhibit the grown of S. mutans by showing a halo of inhibition around the discs. Moreover, this antibacterial activity is enhanced by the addition of silver ion in an amount below to known toxic concentration, showing a synergetic effect that can further potentiate even more these HA nanoparticles. This work demonstrates that solvothermal method is a promising synthesis way for producing antibacterial hydroxyapatites nanoparticles for biomedical applications such as oral tissue regeneration.