Stefano Perni

The antimicrobial properties of light-activated polymers containing methylene blue and gold nanoparticles

We report the formation of polysiloxane polymers containing embedded methylene blue and gold nanoparticles incorporated by a swell-encapsulation-shrink method. These polymers show significant antimicrobial activity against methicillin-resistant Staphylococcus aureus and Escherichia coli with up to a 3.5 log(10) reduction in the viable count when exposed for 5 min to light from a low power 660 nm laser. The bacterial kill is due to the light-induced production of singlet oxygen and other reactive oxygen species by the methylene blue. Interestingly, the presence of 2 nm gold nanoparticles significantly enhanced the ability of the methylene blue to kill bacteria.

Toluidine blue-containing polymers exhibit potent bactericidal activity when irradiated with red laser light

Toluidine blue and toluidine blue-nanogold mixtures were incorporated into polyurethane and silicone polymers by a swell-encapsulation-shrink method using acetone-water mixtures. The surface and mechanical properties of the polymers were changed by the swell-shrink process especially the Youngs modulus, but not by the introduction of toluidine blue or nanogold. The antibacterial properties of the various polymers were assessed under laser irradiation at 634 nm against Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). The toluidine blue-incorporated polymers showed kills of (>105 cfu/ml) for MRSA after just one minute of exposure. This is, to our knowledge, the most potent light-activated antimicrobial polymer combination reported to date.

A novel bone cement impregnated with silver–tiopronin nanoparticles: its antimicrobial, cytotoxic, and mechanical properties

Post-operatory infections in orthopedic surgeries pose a significant risk. The common approach of using antibiotics, both parenterally or embedded in bone cement (when this is employed during surgery) faces the challenge of the rising population of pathogens exhibiting resistance properties against one or more of these compounds; therefore, novel approaches need to be developed. Silver nanoparticles appear to be an exciting prospect because of their antimicrobial activity and safety at the levels used in medical applications. In this paper, a novel type of silver nanoparticles capped with tiopronin is presented. Two ratios of reagents during synthesis were tested and the effect on the nanoparticles investigated through TEM, TGA, and UV-Vis spectroscopy. Once encapsulated in bone cement, only the nanoparticles with the highest amount of inorganic fraction conferred antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) at concentrations as low as 0.1% w/w. No other characteristics of the bone cement, such as cytotoxicity or mechanical properties, were affected by the presence of the nanoparticles. Our work presents a new type of silver nanoparticles and demonstrates that they can be embedded in bone cement to prevent infections once the synthetic conditions are tailored for such applications.

Antimicrobial activity of methylene blue and toluidine blue O covalently bound to a modified silicone polymer surface

Methylene Blue or Toluidine Blue O were covalently bound to an activated silicone polymer by means of an amide condensation reaction. UV-visible absorption spectra confirmed that the dye was surface bound. The new polymers with covalently attached dye display significant bactericidal activity against Escherichia coli and Staphylococcus epidermidis with a 99.999% reduction in viable bacteria after four minutes exposure to a low power laser.

Multiasperity contact adhesion model for universal asperity height and radius of curvature distributions.

A new approach to the multiasperities contact interaction between two surfaces is presented. Each asperity is individually considered with its own different height and radius of curvature. Different materials, such as polyvinylchlorine (PVC) and stainless steel, are used as model systems. For each of the model materials, a set of asperities was generated using Monte Carlo method. Both asperity heights and radii were based on their statistical distributions experimentally obtained. Contact forces were determined for each asperity at a given distance between the two surfaces, while the deformation of each asperity was calculated according to the Johnson-Kendall-Roberts (JKR) or the Derjaguin-Muller-Toporov (DMT) model (depending on the material). The contribution of each asperity to the overall surface was summed, and the overall contact force was determined. The developed method was validated against contact force measurements obtained with atomic force microscopy (AFM).

Silver nanoparticle based antibacterial methacrylate hydrogels potential for bone graft applications

Infections are frequent and very undesired occurrences after orthopedic procedures; furthermore, the growing concern caused by the rise in antibiotic resistance is progressively dwindling the efficacy of such drugs. Artificial bone graft materials could solve some of the problems associated with the gold standard use of natural bone graft such as limited bone material, pain at the donor site and rejections if donor tissue is used. We have previously described new acrylate base nanocomposite hydrogels as bone graft materials. In the present paper, we describe the integration of silver nanoparticles in the polymeric mineralized biomaterial to provide non-antibiotic antibacterial activity against Staphylococcus epidermidis and Methicillin-resistant Staphylococcus aureus. Two different crosslinking degrees were tested and the silver nanoparticles were integrated into the composite matrix by means of three different methods: entrapment in the polymeric hydrogel before the mineralization; diffusion during the process of calcium phosphate crystallization and adsorption post-mineralization. The latter being generally the most effective method of encapsulation; however, the adsorption of silver nanoparticles inside the pores of the biomaterial led to a decreasing antibacterial activity for adsorption time longer than 2 days.

Incorporation of methylene blue and nanogold into polyvinyl chloride catheters; a new approach for light-activated disinfection of surfaces

Methylene blue and 2 nm gold nanoparticles were incorporated into commercial PVC catheters by use of a simple “swell–encapsulation–shrink” method using acetone–water mixtures. Neither the methylene blue nor the nanogold leached into aqueous solution and the assemblage was stable to photodegradation upon laser irradiation. Exposure of the modified catheters to red laser light for 4–8 minutes induced the lethal photosensitisation of Staphylococcus epidermidis and Escherichia coli. Results from time-resolved EPR spectroscopy suggested that enhanced methylene blue triplet state production occurs in the presence of 2 nm gold nanoparticles. The implication being that the levels of reactive oxygen species are higher in these co-doped materials than with methylene blue alone.

Surface roughness mediated adhesion forces between borosilicate glass and gram-positive bacteria

It is well-known that a number of surface characteristics affect the extent of adhesion between two adjacent materials. One of such parameters is the surface roughness as surface asperities at the nanoscale level govern the overall adhesive forces. For example, the extent of bacterial adhesion is determined by the surface topography; also, once a bacteria colonizes a surface, proliferation of that species will take place and a biofilm may form, increasing the resistance of bacterial cells to removal. In this study, borosilicate glass was employed with varying surface roughness and coated with bovine serum albumin (BSA) in order to replicate the protein layer that covers orthopedic devices on implantation. As roughness is a scale-dependent process, relevant scan areas were analyzed using atomic force microscope (AFM) to determine Ra; furthermore, appropriate bacterial species were attached to the tip to measure the adhesion forces between cells and substrates. The bacterial species chosen (Staphylococci and Streptococci) are common pathogens associated with a number of implant related infections that are detrimental to the biomedical devices and patients. Correlation between adhesion forces and surface roughness (Ra) was generally better when the surface roughness was measured through scanned areas with size (2 × 2 μm) comparable to bacteria cells. Furthermore, the BSA coating altered the surface roughness without correlation with the initial values of such parameter; therefore, better correlations were found between adhesion forces and BSA-coated surfaces when actual surface roughness was used instead of the initial (nominal) values. It was also found that BSA induced a more hydrophilic and electron donor characteristic to the surfaces; in agreement with increasing adhesion forces of hydrophilic bacteria (as determined through microbial adhesion to solvents test) on BSA-coated substrates.

Success and failure of colloidal approaches in adhesion of microorganisms to surfaces

Biofilms are communities of cells attached to surfaces, their contributions to biological process may be either a benefit or a threat depending on the microorganism involved and on the type of substrate and environment. Biofilm formation is a complex series of steps; due to the size of microorganisms, the initial phase of biofilm formation, the bacterial adhesion to the surface, has been studied and modeled using theories developed in colloidal science. In this review the application of approaches such as Derjaguin, Landau, Verwey, Overbeek (DLVO) theory and its extended version (xDLVO), to bacterial adhesion is described along with the suitability and applicability of such approaches to the investigation of the interface phenomena regulating cells adhesion. A further refinement of the xDLVO theory encompassing the brush model is also discussed. Finally, the evidences of phenomena neglected in colloidal approaches, such as surface heterogeneity and fluid flow, likely to be the source of failure are defined.

In vitro growth factor-induced bio engineering of mature articular cartilage

Articular cartilage maturation is the postnatal development process that adapts joint surfaces to their site-specific biomechanical demands. Maturation involves gross morphological changes that occur through a process of synchronised growth and resorption of cartilage and generally ends at sexual maturity. The inability to induce maturation in biomaterial constructs designed for cartilage repair has been cited as a major cause for their failure in producing persistent cell-based repair of joint lesions. The combination of growth factors FGF2 and TGFβ1 induces accelerated articular cartilage maturation in vitro such that many molecular and morphological characteristics of tissue maturation are observable. We hypothesised that experimental growth factor-induced maturation of immature cartilage would result in a biophysical and biochemical composition consistent with a mature phenotype. Using native immature and mature cartilage as reference, we observed that growth factor-treated immature cartilages displayed increased nano-compressive stiffness, decreased surface adhesion, decreased water content, increased collagen content and smoother surfaces, correlating with a convergence to the mature cartilage phenotype. Furthermore, increased gene expression of surface structural protein collagen type I in growth factor-treated explants compared to reference cartilages demonstrates that they are still in the dynamic phase of the postnatal developmental transition. These data provide a basis for understanding the regulation of postnatal maturation of articular cartilage and the application of growth factor-induced maturation in vitro and in vivo in order to repair and regenerate cartilage defects.