The antibacterial properties of branched peptides based on poly(l-arginine): In vitro antibacterial evaluation and molecular dynamic simulations.

The emergence of bacterial strains resistant to antibiotics is a major issue in the medical field. Antimicrobial peptides are widely studied as they do not generate as much resistant bacterial strains as conventional antibiotics and present a broad range of activity. Among them, the homopolypeptide poly(l-arginine) presents promising antibacterial properties, especially in the perspective of its use in biomaterials. Linear poly(l-arginine) has been extensively studied but the impact of its 3D structure remains unknown. In this study, the antibacterial properties of newly synthesized branched poly(l-arginine) peptides, belonging to the family of multiple antigenic peptides, are evaluated. First, in vitro activities of the peptides shows that branched poly(l-arginine) is more efficient than linear poly(l-arginine) containing the same number of arginine residues. Surprisingly, peptides with more arms and more residues are not the most effective. To better understand these unexpected results, interactions between these peptides and the membranes of Gram positive and Gram negative bacteria are simulated thanks to molecular dynamic. It is observed that the bacterial membrane is more distorted by the branched structure than by the linear one and by peptides containing smaller arms. This mechanism of action is in full agreement with in vitro results and suggest that our simulations form a robust model to evaluate peptide efficiency towards pathogenic bacteria.

Mechanistic Insights into Hyaluronic Acid Induced Peptide Nanofiber Organization in Supramolecular Hydrogels.

Composite hydrogels composed of low-molecular-weight peptide self-assemblies and polysaccharides are gaining great interest as new types of biomaterials. Interactions between polysaccharides and peptide self-assemblies are well reported, but a molecular picture of their impact on the resulting material is still missing. Using the phosphorylated tripeptide precursor Fmoc-FFpY (Fmoc, fluorenylmethyloxycarbonyl; F, phenylalanine; Y, tyrosine; p, phosphate group), we investigated how hyaluronic acid (HA) influences the enzyme-assisted self-assembly of Fmoc-FFY generated in situ in the presence of alkaline phosphatase (AP). In the absence of HA, Fmoc-FFY peptides are known to self-assemble in nanometer thick and micrometer long fibers. The presence of HA leads to the spontaneous formation of bundles of several micrometers thickness. Using fluorescence recovery after photobleaching (FRAP), we find that in the bundles both (i) HA colocalizes with the peptide self-assemblies and (ii) its presence in the bundles is highly dynamic. The attractive interaction between negatively charged peptide fibers and negatively charged HA chains is explained through molecular dynamic simulations that show the existence of hydrogen bonds. Whereas the Fmoc-FFY peptide self-assembly itself is not affected by the presence of HA, this polysaccharide organizes the peptide nanofibers in a nematic phase visible by small-angle X-ray scattering (SAXS). The mean distance d between the nanofibers decreases by increasing the HA concentration c, but remains always larger than the diameter of the peptide nanofibers, indicating that they do not interact directly with each other. At a high enough HA concentration, the nematic organization transforms into an ordered 2D hexagonal columnar phase with a nanofiber distance d of 117 Å. Depletion interaction generated by the polysaccharides can explain the experimental power law variation d∼c-1/4 and is responsible for the bundle formation and organization. Such behavior is thus suggested for the first time on nano-objects using polymers partially adsorbing on self-assembled peptide nanofibers.

Transition from continuous to microglobular shaped peptide assemblies through a Liesegang-like enzyme-assisted mechanism.

Enzyme-assisted self-assembly confined within host materials leads to Liesegang-like spatial structuration when precursor peptides are diffusing through an enzyme-functionalized hydrogel. It is shown here that playing on peptide and enzyme concentrations results in a transition from continuous self-assembled peptide areas to individual microglobules. Their morphology, location, size and buildup mechanism are described. Additionally, it is also found that the enzymes adsorb onto the peptide self-assemblies leading to co-localization of peptide self-assembled microglobules and enzymes. Finally, we find that large microglobules grow at the expense of smaller ones present in their vicinity in a kind of Ostwald ripening process, illustrating the dynamic nature of the peptide self-assembly process within host hydrogels.

Operator versus material influence on film thickness using adhesive resin cement or pre-heated resin composite.

OBJECTIVE: There is a growing interest in using pre-heated composites instead of dual-cured cements when luting indirect restorations. This study evaluated the film thickness obtained from two pre-heated composites and two resin cements, by two different operators. The influence of the materials and the level of expertise of the operator were analyzed. MATERIALS AND METHODS: Forty specimens of human dentin and composite discs were prepared and divided into four groups depending on the luting process. Each group was randomly equally divided to be handled by two operators with different levels of experience. Two of the initial four groups were luted using dual-cured cements and the two remaining groups using light-cured pre-heated composites. Specimen discs were cut after luting, and film thickness was measured using a Digital microscope. Data were analyzed using a 2-way ANOVA with the Holm-Sidak pairwise multiple comparison procedure (p < 0.05). RESULTS: Mean film thickness ranged from 156.16 ± 4.7 to 33.82 ± 0.7 µm. Significant differences (p < 0.001) were noticed between expert and novice results with pre-heated composites. CONCLUSION: Within the limits of this study, using pre-heated composites as a luting cement requires a better level of expertise to achieve a clinically acceptable film thickness. CLINICAL SIGNIFICANCE: Using pre-heated composites as luting agent for indirect restorations requires an experimented skill level to achieve a clinically recommended film thickness.

Escherichia coli Biofilm Formation, Motion and Protein Patterns on Hyaluronic Acid and Polydimethylsiloxane Depend on Surface Stiffness.

The surface stiffness of the microenvironment is a mechanical signal regulating biofilm growth without the risks associated with the use of bioactive agents. However, the mechanisms determining the expansion or prevention of biofilm growth on soft and stiff substrates are largely unknown. To answer this question, we used PDMS (polydimethylsiloxane, 9-574 kPa) and HA (hyaluronic acid gels, 44 Pa-2 kPa) differing in their hydration. We showed that the softest HA inhibited Escherichia coli biofilm growth, while the stiffest PDMS activated it. The bacterial mechanical environment significantly regulated the MscS mechanosensitive channel in higher abundance on the least colonized HA-44Pa, while Type-1 pili (FimA) showed regulation in higher abundance on the most colonized PDMS-9kPa. Type-1 pili regulated the free motion (the capacity of bacteria to move far from their initial position) necessary for biofilm growth independent of the substrate surface stiffness. In contrast, the total length travelled by the bacteria (diffusion coefficient) varied positively with the surface stiffness but not with the biofilm growth. The softest, hydrated HA, the least colonized surface, revealed the least diffusive and the least free-moving bacteria. Finally, this shows that customizing the surface elasticity and hydration, together, is an efficient means of affecting the bacteria's mobility and attachment to the surface and thus designing biomedical surfaces to prevent biofilm growth.

Non-monotonous enzyme-assisted self-assembly profiles resulting from reaction-diffusion processes in host gels.

Reaction-diffusion (RD) processes are responsible for surface and in-depth micropatterning in inanimate and living matter. Here we show that enzyme-assisted self-assembly (EASA) of peptides is a valuable tool to functionnalize host gels. By using a phosphatase distributed in a host hydrogel, the diffusion of phosphorylated peptides from a liquid/host gel interface leads to the spontaneous formation of a pattern of dephosphorylated peptide self-assembly presenting at least two self-assembly maxima. Variation of enzyme and peptide concentrations change the pattern characteristics. When a peptide drop is deposited on a phosphatase functionalized gel, a self-assembly pattern is also formed both along the gel-solution interface and perpendicular to the interface. This self-assembly pattern induces a local change of the gel mechanical properties measured by nanoindentation. Its appearance relies on the formation of self-assembled structures by nucleation and growth processes which are static in the hydrogel. This process presents great similarities with the Liesegang pattern formation and must be taken into account for the functionalization of hydrogels by EASA. A mechanism based on RD is proposed leading to an effective mathematical model accounting for the pattern formation. This work highlights EASA as a tool to design organic Liesegang-like microstructured materials with potential applications in biomaterials and artificial living systems design.

Prediction of coating thickness for polyelectrolyte multilayers via machine learning.

Layer-by-layer (LbL) deposition method of polyelectrolytes is a versatile way of developing functional nanoscale coatings. Even though the mechanisms of LbL film development are well-established, currently there are no predictive models that can link film components with their final properties. The current health crisis has shown the importance of accelerated development of biomedical solutions such as antiviral coatings, and the implementation of machine learning methodologies for coating development can enable achieving this. In this work, using literature data and newly generated experimental results, we first analyzed the relative impact of 23 coating parameters on the coating thickness. Next, a predictive model has been developed using aforementioned parameters and molecular descriptors of polymers from the DeepChem library. Model performance was limited because of insufficient number of data points in the training set, due to the scarce availability of data in the literature. Despite this limitation, we demonstrate, for the first time, utilization of machine learning for prediction of LbL coating properties. It can decrease the time necessary to obtain functional coating with desired properties, as well as decrease experimental costs and enable the fast first response to crisis situations (such as pandemics) where coatings can positively contribute. Besides coating thickness, which was selected as an output value in this study, machine learning approach can be potentially used to predict functional properties of multilayer coatings, e.g. biocompatibility, cell adhesive, antibacterial, antiviral or anti-inflammatory properties.

Effect of the Buffer on the Buildup and Stability of Tannic Acid/Collagen Multilayer Films Applied as Antibacterial Coatings.

The deposition of polyelectrolyte multilayers, obtained by the layer-by-layer (LbL) method, is a well-established technology to design biocompatible and antibacterial coatings aimed at preventing implant-associated infections. Several types of LbL films have been reported to exhibit antiadhesive and/or antibacterial (contact-killing or release-killing) properties governed not only by the incorporated compounds but also by their buildup conditions or their postbuildup treatments. Tannic acid (TA), a natural polyphenol, is known to inhibit the growth of several bacterial strains. In this work, we developed TA/collagen (TA/COL) LbL films built in acetate or citrate buffers at pH 4. Surprisingly, the used buffer impacts not only the physicochemical but also the antibacterial properties of the films. When incubated in physiological conditions, both types of TA/COL films released almost the same amount of TA depending on the last layer and showed an antibacterial effect against Staphylococcus aureus only for citrate-built films. Because of their granular topography, TA/COL citrate films exhibited an efficient release-killing effect with no cytotoxicity toward human gingival fibroblasts. Emphasis is put on a comprehensive evaluation of the physicochemical parameters driving the buildup and the antibacterial property of citrate films. Specifically, complexation strengths between TA and COL are different in the presence of the two buffers affecting the LbL deposition. This work constitutes an important step toward the use of polyphenols as an antibacterial agent when incorporated in LbL films.

Polyanionic Hydrogels as Reservoirs for Polycationic Antibiotic Substitutes Providing Prolonged Antibacterial Activity.

Implantation of biomedical devices is often followed by bacterial infections that may seriously affect implant functionalities and lead to their failure. In the context of bacterial resistance to antibiotics, which is a growing problem worldwide, new strategies that are able to overcome these problems are needed. In this work, we introduce a new formulation of hyaluronic acid (HA)-based antimicrobial material: HA hydrogels loaded with polyarginine (PAR), a polycationic antibiotic substitute. The loading is possible through electrostatic interactions between negatively charged HA and positively charged PAR. Such hydrogels absorb high quantities of PAR, which are then gradually released from the hydrogel. This original system provides a long-lasting antibacterial effect on an in vitro model of repetitive infection, thus demonstrating a strong potential to fight multiple rounds of infections that are resistant to antibiotic treatment. In addition, HA-PAR hydrogels could be deposited onto/into medical devices such as wound dressings and mesh prostheses used in clinical applications. Finally, we performed first in vivo tests of hydrogel-coated mesh materials to verify their biocompatibility in a rat model, which show no difference between control HA hydrogel and PAR-loaded hydrogel in terms of inflammation.

A comparative in vivo study of new shade matching procedures.

AIM: The aim of the present study was to compare six different methods of in vivo color matching: visual shade matching (3D-Master Linearguide shade guide) performed by 1) a novice practitioner, 2) an expert practitioner, 3) the new Rayplicker spectrometer, 4) the Trios III intraoral scanner, and 5) the Omnicam intraoral scanner compared with 6) the Easyshade V spectrophotometer, which was considered as the reference. MATERIALS AND METHODS: Color matching was performed using the 3D-Master references on the sound maxillary right central incisors of 40 subjects. The study first compared the number of colors found using each of the six methods. The references were then converted to the Commission Internationale de l'Eclairage (CIE) L*a*b* values, from which the difference ?E between either two methods ?was derived. Finally, the L* value was used to compare the luminosity measured by each of the six methods. RESULTS: The Rayplicker showed the smallest ?E compared with the Easyshade V. The expert found a closer color to the Easyshade V than did the novice, and both were closer to the Easyshade V than the two intraoral scanners. The intraoral scanners showed notable differences compared with the Easyshade V. The intraoral scanners also offered a reduced choice of colors and recorded the highest luminosities compared with the other methods. CONCLUSION: Within the limitations of this study, the color matching by the Rayplicker was closest to that of the Easyshade V. The good performance of this new device means that it is a challenging competitor for the Easyshade V. Finally, the new methods based on intraoral scanners were less reliable than the spectrophotometers and the visual shade matching.

Photoresponsive Nanometer-Scale Iron Alginate Hydrogels: A Study of Gel-Sol Transition Using a Quartz Crystal Microbalance.

Alginate/Fe3+ hydrogels were fabricated on hyaluronic acid (HA) and poly(allylamine hydrochloride) (PAH) multilayers to yield photoresponsive nanometer-scale hydrogels. Light irradiation of the resulting hydrogels induced the photoreduction of "hard" Fe3+ to "soft" Fe2+ cations, leading to changes in the mechanical properties of the hydrogels related to their cross-linking behavior. The buildup and the phototriggered response of the supported alginate hydrogels were followed in situ with a quartz crystal microbalance (QCM) using an open cell allowing light irradiation from an LED source on top of the hydrogel. The results were correlated to the release profiles of folic acid, employed herein as a drug model, obtained from light-irradiated supported iron alginate hydrogels.

A Pilot Study on Continuous Infusion of 4% Albumin in Critically Ill Patients: Impact on Nosocomial Infection via a Reduction Mechanism for Oxidized Substrates.

Care-related infections affect up to 11% of ICU patients. Running therapeutic albumin is sometimes associated to less infection: whether a specific method of its infusion is of any interest to modulate innate defense is unknown. Our objectives were: 1) to test whether the method for albumin infusion is important to prevent care-related infections and 2) to analyze in vitro the antioxidative role of albumin on host defense proteins during shock (using vasostatin-I as an example). DESIGN: In a prospective, randomized, open-label trial, shock patients were allocated to receive either continuously 4% albumin or intermittently 20% albumin, as long as they were infused with norepinephrine. A translational study including in vivo and in vitro analyses of albumin-vasostatin-I interactions is reported. SETTING: A tertiary ICU caring for 1,000 patients per year. PATIENTS: Fifty shock patients with serum albumin less than 20 g/L. INTERVENTIONS: In vivo colonization and nosocomial infections were recorded and time-dependent changes in serum albumin, chromogranin A, and vasostatin-I concentrations as well. In vitro, we studied biochemical albumin-vasostatin-I relationship using biochemical methods. MEASUREMENTS AND MAIN RESULTS: Over 18 days, we recorded a decrease in colonization (four vs 12 episodes; p = 0.035) and nosocomial infection frequency (two vs 13 episodes; p = 0.002) in patients infused continuously 4% albumin versus controls. In vitro, albumin interacts with the disulfide loop vasostatin-I (residues 17-40) and continuous 4% albumin infusion restores its oxidative status required for antimicrobial activity. CONCLUSIONS: Continuous 4% albumin is effective in reducing care-related infections in shock patients by increasing the availability of antimicrobial vasostatin-I. This might guide future care of shock patients.

Chromatin de-condensation by switching substrate elasticity.

Mechanical properties of the cellular environment are known to influence cell fate. Chromatin de-condensation appears as an early event in cell reprogramming. Whereas the ratio of euchromatin versus heterochromatin can be increased chemically, we report herein for the first time that the ratio can also be increased by purely changing the mechanical properties of the microenvironment by successive 24 h-contact of the cells on a soft substrate alternated with relocation and growth for 7 days on a hard substrate. An initial contact with soft substrate caused massive SW480 cancer cell death by necrosis, whereas approximately 7% of the cells did survived exhibiting a high level of condensed chromatin (21% heterochromatin). However, four consecutive hard/soft cycles elicited a strong chromatin de-condensation (6% heterochromatin) correlating with an increase of cellular survival (approximately 90%). Furthermore, cell survival appeared to be reversible, indicative of an adaptive process rather than an irreversible gene mutation(s). This adaptation process is associated with modifications in gene expression patterns. A completely new approach for chromatin de-condensation, based only on mechanical properties of the microenvironment, without any drug mediation is presented.

Control of Surface-Localized, Enzyme-Assisted Self-Assembly of Peptides through Catalyzed Oligomerization.

Localized self-assembly allowing both spatial and temporal control over the assembly process is essential in many biological systems. This can be achieved through localized enzyme-assisted self-assembly (LEASA), also called enzyme-instructed self-assembly, where enzymes present on a substrate catalyze a reaction that transforms noninteracting species into self-assembling ones. Very few LEASA systems have been reported so far, and the control of the self-assembly process through the surface properties represents one essential step toward their use, for example, in artificial cell mimicry. Here, we describe a new type of LEASA system based on α-chymotrypsin adsorbed on a surface, which catalyzes the production of (KL)nOEt oligopeptides from a KLOEt (K: lysine; L: leucine; OEt ethyl ester) solution. When a critical concentration of the formed oligopeptides is reached near the surface, they self-assemble into ß-sheets resulting in a fibrillar network localized at the interface that can extend over several micrometers. One significant feature of this process is the existence of a lag time before the self-assembly process starts. We investigate, in particular, the effect of the α-chymotrypsin surface density and KLOEt concentration on the self-assembly kinetics. We find that the lag time can be finely tuned through the surface density in α-chymotrypsin and KLOEt concentration. For a given surface enzyme concentration, a critical KLOEt concentration exists below which no self-assembly takes place. This concentration increases when the surface density in enzyme decreases.

Computed Tomographic Study of the Pediatric Diaphragmatic Growth: Application to the Treatment of Congenital Diaphragmatic Hernia.

Background The prosthesis commonly used for the treatment of congenital diaphragmatic hernia (CDH) lacks elasticity to replace the diaphragm's mechanical properties and does not follow the natural growth of the child treated. Objective To determine the appropriate properties required for the prostheses, a CT study on healthy patients was conducted. Methods Two methods of diaphragmatic surface analysis are assessed: the diaphragmatic surface is either estimated using surface 2D estimations (method 1), or calculated using length measures on thoracoabdominal CT scans from children (method 2). Patients are divided into two groups depending on their age: group 1: n = 9; median age: 2.0 months (0.1-9.5); group 2: n = 9; median age: 182.6 months (158.5-235.5). Growth factor between the two groups is calculated and the two methods are statistically compared. Results The ratio group 2/group 1 of the diaphragmatic surfaces was 4.3 ± 0.2 on the left side and 4.0 ± 0.2 on the right side for method 1, and 5.1 ± 0.2 on the left side and 5.1 ± 0.3 on the right side for method 2. The difference in the median values between both methods is statistically significant for both the left and right sides (p = 0.022 and p = 0.002, respectively). Hence, the two methods cannot be used exchangeably. Conclusion The treatment of CDH with large defect remains a challenge because of the high incidence of hernia recurrence probably due to prosthesis defect; thus it is important to estimate the diaphragmatic surface precisely. We aim to develop a prosthesis material that can be commonly used and found a mean diaphragmatic growth factor of approximately 4 to 5 from early childhood to adolescence.

Stretch-Induced Helical Conformations in Poly(l-lysine)/Hyaluronic Acid Multilayers.

We investigate the effect of stretching on the secondary structure of cross-linked poly(l-lysine)/hyaluronic acid (PLL/HA) multilayers. We show that stretching these films induces changes in the secondary structure of PLL chains. Our results suggest that not only α- but also 310-helices might form in the film under stretching. Such 310-helices have never been observed for PLL so far. These changes of the secondary structure of PLL are reversible, i.e., when returning to the nonstretched state one recovers the initial film structure. Using molecular dynamics simulations of chains composed of 20 l-lysine residues (PLL20), we find that these chains never adopt a helical conformation in water. In contrast, when the end-to-end distance of the chains is restrained to values smaller than the mean end-to-end distance of free chains, a distance domain rarely explored by the free chains, helical conformations become accessible. Moreover, the formation of not only α- but also 310-helices is predicted by the simulations. These results suggest that the change of the end-to-end distance of PLL chains in the stretched film is at the origin of the helix formation.

Film Self-Assembly of Oppositely Charged Macromolecules Triggered by Electrochemistry through a Morphogenic Approach.

The development of new surface functionalization methods that are easy to use, versatile, and allow local deposition represents a real scientific challenge. Overcoming this challenge, we present here a one-pot process that consists in self-assembling, by electrochemistry on an electrode, films made of oppositely charged macromolecules. This method relies on a charge-shifting polyanion, dimethylmaleic-modified poly(allylamine) (PAHd), that undergoes hydrolysis at acidic pH, leading to an overall switching of its charge. When a mixture of the two polyanions, PAHd and poly(styrenesulfonate) (PSS), is placed in contact with an electrode, where the pH is decreased locally by electrochemistry, the transformation of PAHd into a polycation (PAH) leads to the continuous self-assembly of a nanometric PAH/PSS film by electrostatic interactions. The pH decrease is obtained by the electrochemical oxidation of hydroquinone, which produces protons locally over nanometric distances. Using a negatively charged enzyme, alkaline phosphatase (AP), instead of PSS, this one-pot process allows the creation of enzymatically active films. Under mild conditions, self-assembled PAH/AP films have an enzymatic activity which is adjustable simply by controlling the self-assembly time. The selective functionalization of microelectrode arrays by PAH/AP was achieved, opening the route toward miniaturized biosensors.

Bioactive Seed Layer for Surface-Confined Self-Assembly of Peptides.

The design and control of molecular systems that self-assemble spontaneously and exclusively at or near an interface represents a real scientific challenge. We present here a new concept, an active seed layer that allows to overcome this challenge. It is based on enzyme-assisted self-assembly. An enzyme, alkaline phosphatase, which transforms an original peptide, Fmoc-FFY(PO4 (2-) ), into an efficient gelation agent by dephosphorylation, is embedded in a polyelectrolyte multilayer and constitutes the "reaction motor". A seed layer composed of a polyelectrolyte covalently modified by anchoring hydrogelator peptides constitutes the top of the multilayer. This layer is the nucleation site for the Fmoc-FFY peptide self-assembly. When such a film is brought in contact with a Fmoc-FFY(PO4 (2-) ) solution, a nanofiber network starts to form almost instantaneously which extents up to several micrometers into the solution after several hours. We demonstrate that the active seed layer allows convenient control over the self-assembly kinetics and the geometric features of the fiber network simply by changing its peptide density.

A new biomimetic route to engineer enzymatically active mechano-responsive materials.

Using modified ß-galactosidase covalently linked to cross-linked polyelectrolyte multilayers (PEM), catalytically active materials have been designed. Their enzymatic activity can be modulated, partially in a reversible way, simply by stretching. This strategy, based on enzyme conformational changes, constitutes a new tool for the development of biocatalytic mechano-responsive materials.

Cell Alignment Driven by Mechanically Induced Collagen Fiber Alignment in Collagen/Alginate Coatings.

For many years it has been a major challenge to regenerate damaged tissues using synthetic or natural materials. To favor the healing processes after tendon, cornea, muscle, or brain injuries, aligned collagen-based architectures are of utmost interest. In this study, we define a novel aligned coating based on a collagen/alginate (COL/ALG) multilayer film. The coating exhibiting a nanofibrillar structure is cross-linked with genipin for stability in physiological conditions. By stretching COL/ALG-coated polydimethylsiloxane substrates, we developed a versatile method to align the collagen fibrils of the polymeric coating. Assays on cell morphology and alignment were performed to investigate the properties of these films. Microscopic assessments revealed that cells align with the stretched collagen fibrils of the coating. The degree of alignment is tuned by the stretching rate (i.e., the strain) of the COL/ALG-coated elastic substrate. Such coatings are of great interest for strategies that require aligned nanofibrillar biological material as a substrate for tissue engineering.