Easy, Flexible and Standardizable Anti-Nascent Biofilm Activity Assay to Assess Implant Materials.

Medical implants have improved the quality of life of many patients. However, surgical intervention may eventually lead to implant microbial contamination. The aims of this research were to develop an easy, robust, quantitative assay to assess surface antimicrobial activities, especially the anti-nascent biofilm activity, and to identify control surfaces, allowing for international comparisons. Using new antimicrobial assays to assess the inhibition of nascent biofilm during persistent contact or after transient contact with bacteria, we show that the 5 cent Euro coin or other metal-based antibacterial coins can be used as positive controls, as more than 4 log reduction on bacterial survival was observed when using either S. aureus or P. aeruginosa as targets. The methods and controls described here could be useful to develop an easy, flexible and standardizable assay to assess relevant antimicrobial activities of new implant materials developed by industries and academics.

Two compartmental fractional derivative model with general fractional derivative.

This study presents a new two compartmental model with, recently defined General fractional derivative. We review that concept of General fractional derivative and use the kernel function that generalizes the classical Caputo derivative in a mathematically consistent way. Next we use this model to study the release of antibiotic gentamicin in poly (vinyl alcohol)/gentamicin(PVA/Gent) hydrogel aimed for wound dressing in medical treatment of deep chronical wounds. The PVA/Gent hydrogel was prepared by physical cross linking of poly (vinyl alcohol) dispersion using freezing-thawing method, and then was swollen in gentamicin solution at 37 °C during 48 h. The concentration of released gentamicin was determined using a high-performance liquid chromatography coupled with mass spectrometer. The advantage of this model is the existence of new parameters in the definition of fractional derivative, as compared with classical fractional compartmental models. The model proposed here in the special case reduces to the classical (integer order) linear two compartmental model as well as classical fractional order two compartmental model since it has more parameters that are determined from the experimental results.

Macrophages, the main marker in biocompatibility evaluation of new hydrogels after subcutaneous implantation in rats.

Biocompatibility of materials is one of the most important conditions for their successful application in tissue regeneration and repair. Cell-surface interactions stimulate adhesion and activation of macrophages whose acquaintance can assist in designing novel biomaterials that promote favorable macrophage-biomaterial surface interactions for clinical application. This study is designed to determine the distribution and number of macrophages as a means of biocompatibility evaluation of two newly synthesized materials [silver/poly(vinyl alcohol) (Ag/PVA) and silver/poly(vinyl alcohol)/graphene (Ag/PVA/Gr) nanocomposite hydrogels] in vivo, with approval of the Ethics Committee of the Faculty of Veterinary Medicine, University of Belgrade. Macrophages and giant cells were analyzed in tissue sections stained by routine H&E and immunohistochemical methods (CD68+). Statistical relevance was determined in the statistical software package SPSS 20 (IBM corp). The results of the study in terms of the number of giant cells localized around the implant showed that their number was highest on the seventh postoperative day (p.o.d.) in the group implanted with Ag/PVA hydrogels, and on the 30th p.o.d. in the group implanted with Ag/PVA/Gr. Interestingly, the number of macrophages measured in the capsular and pericapsular space was highest in the group implanted with the commercial Suprasorb© material. The increased macrophage number, registered around the Ag/PVA/Gr implant on 60th p.o.d. indicates that the addition of graphene can, in a specific way, modulate different biological responses of tissues in the process of wound healing, regeneration, and integration.

Electrophoretic Deposition of Biocompatible and Bioactive Hydroxyapatite-Based Coatings on Titanium.

Current trends in biomaterials science address the issue of integrating artificial materials as orthopedic or dental implants with biological materials, e.g., patients' bone tissue. Problems arise due to the simple fact that any surface that promotes biointegration and facilitates osteointegration may also provide a good platform for the rapid growth of bacterial colonies. Infected implant surfaces easily lead to biofilm formation that poses a major healthcare concern since it could have destructive effects and ultimately endanger the patients' life. As of late, research has centered on designing coatings that would eliminate possible infection but neglected to aid bone mineralization. Other strategies yielded surfaces that could promote osseointegration but failed to prevent microbial susceptibility. Needless to say, in order to assure prolonged implant functionality, both coating functions are indispensable and should be addressed simultaneously. This review summarizes progress in designing multifunctional implant coatings that serve as carriers of antibacterial agents with the primary intention of inhibiting bacterial growth on the implant-tissue interface, while still promoting osseointegration.

Assessing the Bioactivity of Gentamicin-Preloaded Hydroxyapatite/Chitosan Composite Coating on Titanium Substrate.

The electrophoretic deposition process (EPD) was utilized to produce bioactive hydroxyapatite/chitosan (HAP/CS) and hydroxyapatite/chitosan/gentamicin (HAP/CS/Gent) coatings on titanium. The bioactivity of newly synthesized composite coatings was investigated in the simulated body fluid (SBF) and examined by X-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy. The obtained results revealed carbonate-substituted hydroxyapatite after immersion in SBF, emphasizing the similarity of the biomimetically grown HAP with the naturally occurring apatite in the bone. The formation of biomimetic HAP was confirmed by electrochemical impedance spectroscopy and polarization measurements, through the decrease in corrosion current density and coating capacitance values after 28-day immersion in SBF. The osseointegration ability was further validated by measuring the alkaline phosphatase activity (ALP) indicating the favorable osseopromotive properties of deposited coatings (significant increase in ALP levels for both HAP/CS (3.206 U mL-1) and HAP/CS/Gent (4.039 U mL-1) coatings, compared to the control (0.900 U mL-1)). Drug-release kinetics was investigated in deionized water at 37 °C by high-performance liquid chromatography coupled with mass spectrometry. Release profiles revealed the beneficial "burst-release effect" (∼21% of gentamicin released in the first 48 h) as a potentially promising solution against the biofilm formation in the initial period. When tested against human and mice fibroblast cells (MRC-5 and L929), both composite coatings showed a noncytotoxic effect (viability >85%), providing a promising basis for further medical application trials.

Antibacterial graphene-based hydroxyapatite/chitosan coating with gentamicin for potential applications in bone tissue engineering.

Electrophoretic deposition process (EPD) was successfully used for obtaining graphene (Gr)-reinforced composite coating based on hydroxyapatite (HAP), chitosan (CS), and antibiotic gentamicin (Gent), from aqueous suspension. The deposition process was performed as a single step process at a constant voltage (5 V, deposition time 12 min) on pure titanium foils. The influence of graphene was examined through detailed physicochemical and biological characterization. Fourier transform infrared spectroscopy, field emission scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, Raman, and X-ray photoelectron analyses confirmed the formation of composite HAP/CS/Gr and HAP/CS/Gr/Gent coatings on Ti. Obtained coatings had porous, uniform, fracture-free surfaces, suggesting strong interfacial interaction between HAP, CS, and Gr. Large specific area of graphene enabled strong bonding with chitosan, acting as nanofiller throughout the polymer matrix. Gentamicin addition strongly improved the antibacterial activity of HAP/CS/Gr/Gent coating that was confirmed by antibacterial activity kinetics in suspension and agar diffusion testing, while results indicated more pronounced antibacterial effect against Staphylococcus aureus (bactericidal, viable cells number reduction >3 logarithmic units) compared to Escherichia coli (bacteriostatic, <3 logarithmic units). MTT assay indicated low cytotoxicity (75% cell viability) against MRC-5 and L929 (70% cell viability) tested cell lines, indicating good biocompatibility of HAP/CS/Gr/Gent coating. Therefore, electrodeposited HAP/CS/Gr/Gent coating on Ti can be considered as a prospective material for bone tissue engineering as a hard tissue implant.

Functional bioreactor characterization to assess potentials of nanocomposites based on different alginate types and silver nanoparticles for use as cartilage tissue implants.

In this work, functional characterization of biomaterials concerning potential application as articular cartilage implants was performed by using a biomimetic bioreactor with dynamic compression in the physiological regime (10% strain, 0.84 Hz frequency, 1 h on/1 h off). Specifically, two alginate types with low (LG) and high (HG) guluronic/mannuronic residue ratios with electrochemically synthesized silver nanoparticles (AgNPs) were evaluated. HG Ag/alginate hydrogels were clearly indicated as potential candidates due to better initial mechanical properties as compared to LG hydrogels (dynamic compression modulus of ~60 vs. ~40 kPa) as well as the mechanical stability displayed during 7 days of dynamic compression. Cytotoxicity studies in 3D bovine cartilage explant cultures under dynamic compression have shown negligible effects as compared to standard 2D monolayers of bovine chondrocytes where moderate cytotoxicity was observed. Finally, experimental and mathematical modeling studies revealed different mechanisms of AgNP release under physiological-like bioreactor conditions as compared to static conditions. Overall, the results clearly demonstrate bioreactor advantages in characterization and selection of candidate biomaterials as well as potentials to bridge the in vitro-in vivo gap. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 755-768, 2019.

Comparative in vivo evaluation of novel formulations based on alginate and silver nanoparticles for wound treatments.

In the present study, possibilities for using novel nanocomposites based on alginate and silver nanoparticles for wound treatment were investigated in a second-degree thermal burn model in Wistar rats. Silver nanoparticles (AgNPs) were electrochemically synthesized in alginate solutions that were further utilized to obtain the Ag/alginate solution and microfibers for subsequent in vivo studies. Daily applications of the Ag/alginate colloid solution, containing AgNPs, alginate and ascorbic acid (G3), wet Ag/alginate microfibers containing AgNPs (G5) and dry Ag/alginate microfibers containing AgNPs (G6) were compared to treatments with a commercial cream containing silver sulfadiazine (G2) and a commercial Ca-alginate wound dressing containing silver ions (G4), as well as to the untreated controls (G1). Results of the in vivo study have shown faster healing in treated wounds, which completely healed on day 19 (G4, G5 and G6) and 21 (G2 and G3) after the thermal injury, while the period for complete reepitelization of untreated wounds (G1) was 25 days. The macroscopic analysis has shown that scabs fell off between day 10 and 12 after the thermal injury induction in treated groups, whereas between day 15 and 16 in the control group. These macroscopic findings were supported by the results of histopathological analyses, which have shown enhanced granulation and reepithelization, reduced inflammation and improved organization of the extracellular matrix in treated groups without adverse effects. Among the treated groups, dressings based on Ca-alginate (G4-G6) induced enhanced healing as compared to the other two groups (G2, G3), which could be attributed to additional stimuli of released Ca2+. The obtained results indicated potentials of novel nanocomposites based on alginate and AgNPs for therapeutic applications in wound treatments.

Gentamicin-Loaded Bioactive Hydroxyapatite/Chitosan Composite Coating Electrodeposited on Titanium.

Composite coating of antibiotic gentamicin (Gent), natural polymer chitosan (CS), and hydroxyapatite (HAP) was successfully assessed by applying the electrophoretic deposition (EPD) technique. EPD was performed under optimized deposition conditions (5 V, 12 min) on pure titanium plates, to obtain HAP/CS and HAP/CS/Gent composite coatings in a single step from three-component aqueous suspension, with favorable antibacterial properties. Composite coatings were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray photoelectron analysis, confirming the formation of composite HAP/CS and HAP/CS/Gent coatings on the titanium surface, which is due to intermolecular hydrogen bonds. Employing the XRD technique, HAP was detected by obtaining the characteristic diffraction maximums. Good antibacterial activity of the composite coating loaded with antibiotic (HAP/CS/Gent) was confirmed against Staphylococcus aureus and Escherichia coli, pointing to the high potential for bioapplication. Introduction of gentamicin in HAP/CS/Gent coating caused very mild cytotoxicity in the tested cell lines MRC-5 and L929. MTT testing was used to evaluate cell viability, and HAP/CS was classified as noncytotoxic.

Corrosion evaluation of zirconium doped oxide coatings on aluminum formed by plasma electrolytic oxidation.

The plasma electrolytic oxidation (PEO) of aluminum in sodium tungstate (Na(2)WO(4) · (2)H(2)O) and Na(2)WO(4) · (2)H(2)O doped with Zr was analyzed in order to obtain oxide coatings with improved corrosion resistance. The influence of current density in PEO process and anodization time was investigated, as well as the influence of Zr, with the aim to find out how they affect the chemical content, morphology, surface roughness, and corrosion stability of oxide coatings. It was shown that the presence of Zr increases the corrosion stability of oxide coatings for all investigated PEO times. Evolution of EIS spectra during the exposure to 3% NaCl, as a strong corrosive agent, indicated the highest corrosion stability for PEO coating formed on aluminum at 70 mA/cm(2) for 2 min in a zirconium containing electrolyte.

A comprehensive approach to in vitro functional evaluation of Ag/alginate nanocomposite hydrogels.

In this work, we present a comprehensive approach to evaluation of alginate microbeads with included silver nanoparticles (AgNPs) at the concentration range of 0.3-5mM for potential biomedical use by combining cytotoxicity, antibacterial activity, and silver release studies. The microbeads were investigated regarding drying and rehydration showing retention of ∼ 80-85% of the initial nanoparticles as determined by UV-vis and SEM analyses. Both wet and dry microbeads were shown to release AgNPs and/or ions inducing similar growth delays of Staphylococcus aureus and Escherichia coli at the total released silver concentrations of ∼ 10 µg/ml. On the other hand, these concentrations were highly toxic for bovine chondrocytes in conventional monolayer cultures while nontoxic when cultured in alginate microbeads under biomimetic conditions in 3D perfusion bioreactors. The applied approach outlined directions for further optimization studies demonstrating Ag/alginate microbeads as potentially attractive components of soft tissue implants as well as antimicrobial wound dressings.

Novel bioactive antimicrobial lignin containing coatings on titanium obtained by electrophoretic deposition.

Hydroxyapatite (HAP) is the most suitable biocompatible material for bone implant coatings; its brittleness, however, is a major obstacle, and the reason why research focuses on creating composites with biopolymers. Organosolv lignin (Lig) is used for the production of composite coatings, and these composites were examined in this study. Titanium substrate is a key biomedical material due to its well-known properties, but infections of the implantation site still impose a serious threat. One approach to prevent infection is to improve antimicrobial properties of the coating material. Silver doped hydroxyapatite (Ag/HAP) and HAP coatings on titanium were obtained by an electrophoretic deposition method in order to control deposited coating mass and morphology by varying applied voltage and deposition time. The effect of lignin on microstructure, morphology and thermal behavior of biocomposite coatings was investigated. The results showed that higher lignin concentrations protect the HAP lattice during sintering, improving coating stability. The corrosion stability was evaluated in simulated body fluid (SBF) at 37 °C. Newly formed plate-shaped carbonate-HAP was detected, indicating enhanced bioactive performance. The antimicrobial efficiency of Ag/HAP/Lig was confirmed by its higher reduction of bacteria Staphylococcus aureus TL (S. aureus TL) than of HAP/Lig coating. Cytotoxicity assay revealed that both coatings can be classified as non-toxic against healthy immunocompetent peripheral blood mononuclear cells (PBMC).

Bioreactor validation and biocompatibility of Ag/poly(N-vinyl-2-pyrrolidone) hydrogel nanocomposites.

Silver/poly(N-vinyl-2-pyrrolidone) (Ag/PVP) nanocomposites containing Ag nanoparticles at different concentrations were synthesized using γ-irradiation. Cytotoxicity of the obtained nanocomposites was determined by MTT assay in monolayer cultures of normal human immunocompetent peripheral blood mononuclear cells (PBMC) that were either non-stimulated or stimulated to proliferate by mitogen phytohemagglutinin (PHA), as well as in human cervix adenocarcinoma cell (HeLa) cultures. Silver release kinetics and mechanical properties of nanocomposites were investigated under bioreactor conditions in the simulated body fluid (SBF) at 37°C. The release of silver was monitored under static conditions, and in two types of bioreactors: perfusion bioreactors and a bioreactor with dynamic compression coupled with SBF perfusion simulating in vivo conditions in articular cartilage. Ag/PVP nanocomposites exhibited slight cytotoxic effects against PBMC at the estimated concentration of 0.4 µmol dm(-3), with negligible variations observed amongst different cell cultures investigated. Studies of the silver release kinetics indicated internal diffusion as the rate limiting step, determined by statistically comparable results obtained at all investigated conditions. However, silver release rate was slightly higher in the bioreactor with dynamic compression coupled with SBF perfusion as compared to the other two systems indicating the influence of dynamic compression. Modelling of silver release kinetics revealed potentials for optimization of Ag/PVP nanocomposites for particular applications as wound dressings or soft tissue implants.

Corrosion stability and bioactivity in simulated body fluid of silver/hydroxyapatite and silver/hydroxyapatite/lignin coatings on titanium obtained by electrophoretic deposition.

Hydroxyapatite is the most suitable biocompatible material for bone implant coatings. However, its brittleness is a major obstacle, and that is why, recently, research focused on creating composites with various biopolymers. In this study, hydroxyapatite coatings were modified with lignin in order to attain corrosion stability and surface porosity that enables osteogenesis. Incorporating silver, well known for its antimicrobial properties, seemed the best strategy for avoiding possible infections. The silver/hydroxyapatite (Ag/HAP) and silver/hydroxyapatite/lignin (Ag/HAP/Lig) coatings were cathaphoretically deposited on titanium from ethanol suspensions, sintered at 900 °C in Ar, and characterized by X-ray diffraction, scanning electron microscopy, field emission scanning electron microscopy, attenuated total reflection Fourier transform infrared, and X-ray photoelectron spectroscopy. The corrosion stability of electrodeposited coatings was evaluated in vitro in Kokubo's simulated body fluid (SBF) at 37 °C using electrochemical impedance spectroscopy. Bioactivity was estimated by immersion in SBF to evaluate the formation of hydroxyapatite on the coating surface. A microcrystalline structure of newly formed plate-shaped carbonate-hydroxyapatite was detected after only 7 days, indicating enhanced bioactive behavior. Both coatings had good corrosion stability during a prolonged immersion time. Among the two, the Ag/HAP/Lig coating had a homogeneous surface, less roughness, and low values of contact angle.

Novel alginate based nanocomposite hydrogels with incorporated silver nanoparticles.

Alginate colloid solution containing electrochemically synthesized silver nanoparticles (AgNPs) was investigated regarding the nanoparticle stabilization and possibilities for production of alginate based nanocomposite hydrogels in different forms. AgNPs were shown to continue to grow in alginate solutions for additional 3 days after the synthesis by aggregative mechanism and Ostwald ripening. Thereafter, the colloid solution remains stable for 30 days and could be used alone or in mixtures with aqueous solutions of poly(vinyl alcohol) (PVA) and poly(N-vinyl-2-pyrrolidone) (PVP) while preserving AgNPs as verified by UV-Vis spectroscopy studies. We have optimized techniques for production of Ag/alginate microbeads and Ag/alginate/PVA beads, which were shown to efficiently release AgNPs decreasing the Escherichia coli concentration in suspensions for 99.9% over 24 h. Furthermore, Ag/hydrogel discs based on alginate, PVA and PVP were produced by freezing-thawing technique allowing adjustments of hydrogel composition and mechanical properties as demonstrated in compression studies performed in a biomimetic bioreactor.

The effect of the addition of colloidal iridium oxide into sol-gel obtained titanium and ruthenium oxide coatings on titanium on their electrochemical properties.

Electrochemical properties of sol-gel processed Ti(0.6)Ir(0.4)O(2) and Ti(0.6)Ru(0.3)Ir(0.1)O(2) coatings on titanium substrate were investigated using cyclic voltammetry, polarization measurements and electrochemical impedance spectroscopy and compared to the properties of Ti(0.6)Ru(0.4)O(2) coating. The role of iridium oxide in the improvement of the electrocatalytic, capacitive and stability properties of titanium anodes activated by a RuO(2)-TiO(2) coating is discussed. The oxide sols were prepared by forced hydrolysis of the metal chlorides. The characterization by dynamic light scattering and X-ray diffraction showed that polydisperse oxide sols were obtained with the particles tending to form agglomerates. The presence of IrO(2) causes a suppression of the X-ray diffraction peaks of TiO(2) and RuO(2) in the sol-gel prepared Ti(0.6)Ir(0.4)O(2) and Ti(0.6)Ru(0.3)Ir(0.1)O(2) coatings. The IrO(2)-containing coatings had an enhanced charge storage ability and activity for the oxygen evolution reaction (OER) in comparison to Ti(0.6)Ru(0.4)O(2) coating. The voltammogram of the Ti(0.6)Ir(0.4)O(2)/Ti electrode showed well-resolved peaks related to Ir redox transitions, which are responsible for the enhanced charge storage ability of IrO(2)-containing coatings. Redox transitions of Ir were also registered in the high-frequency domain of the ac impedance spectra of the coatings as a semicircle with characteristics insensitive to the electrolyte composition and to the electrode potential prior to OER. However, the semicircle characteristics were different for the two IrO(2)-containing coatings, as well as at potentials outside the OER in comparison to those at which the OER occurs.