Toughening 3D printed biomimetic hydroxyapatite scaffolds: Polycaprolactone-based self-hardening inks.

The application of 3D printing to calcium phosphates has opened unprecedented possibilities for the fabrication of personalized bone grafts. However, their biocompatibility and bioactivity are counterbalanced by their high brittleness. In this work we aim at overcoming this problem by developing a self-hardening ink containing reactive ceramic particles in a polycaprolactone solution instead of the traditional approach that use hydrogels as binders. The presence of polycaprolactone preserved the printability of the ink and was compatible with the hydrolysis-based hardening process, despite the absence of water in the ink and its hydrophobicity. The microstructure evolved from a continuous polymeric phase with loose ceramic particles to a continuous network of hydroxyapatite nanocrystals intertwined with the polymer, in a configuration radically different from the polymer/ceramic composites obtained by fused deposition modelling. This resulted in the evolution from a ductile behavior, dominated by the polymer, to a stiffer behavior as the ceramic phase reacted. The polycaprolactone binder provides two highly relevant benefits compared to hydrogel-based inks. First, the handleability and elasticity of the as-printed scaffolds, together with the proven possibility of eliminating the solvent, opens the door to implanting the scaffolds freshly printed once lyophilized, while in a ductile state, and the hardening process to take place inside the body, as in the case of calcium phosphate cements. Second, even with a hydroxyapatite content of more than 92 wt.%, the flexural strength and toughness of the scaffolds after hardening are twice and five times those of the all-ceramic scaffolds obtained with the hydrogel-based inks, respectively. STATEMENT OF SIGNIFICANCE: Overcoming the brittleness of ceramic scaffolds would extend the applicability of synthetic bone grafts to high load-bearing situations. In this work we developed a 3D printing ink by replacing the conventional hydrogel binder with a water-free polycaprolactone solution. The presence of polycaprolactone not only enhanced significantly the strength and toughness of the scaffolds while keeping the proportion of bioactive ceramic phase larger than 90 wt.%, but it also conferred flexibility and manipulability to the as-printed scaffolds. Since they are able to harden upon contact with water under physiological conditions, this opens up the possibility of implanting them immediately after printing, while they are still in a ductile state, with clear advantages for fixation and press-fit in the bone defect.

Engineering alginate-based injectable hydrogels combined with bioactive polymers for targeted plasma-derived oxidative stress delivery in osteosarcoma therapy.

Reactive Oxygen and Nitrogen Species (RONS) in biological systems display hormetic effects, capable of either promoting cell regenerative effects or inducing cell death. Recently, hydrogels have emerged as a promising delivery platform for RONS generated from Cold Atmospheric Plasmas (CAP), known as Plasma-Treated Hydrogels (PTH). PTH have been proposed as an alternative therapy to conventional cancer treatments, offering reduced side effects through the controlled and localized delivery of plasma-derived RONS. In this work, we have developed alginate-based PTH with dual therapeutic action provided by plasma-derived RONS acting as selective anticancer agents for osteosarcoma treatment, and biomolecules (hyaluronic acid and gelatin) to promote stem cell-mediated bone regeneration. For this purpose, we designed a novel manufacturing process to maximize the load of plasma-derived RONS within the PTH. Then, we assessed the PTH bioactivity on osteosarcoma MG-63 cells, and human mesenchymal stem cells (hMSCs). The results showed that the PTH composed of 0.25 % alginate +1 % hyaluronic acid is the most promising formulation in osteosarcoma treatment, showing a dual-action bioactivity as a selective cytotoxic anticancer agent, and as promoter of the proliferation and osteogenic differentiation of hMSCs. These findings provide strong evidence of the significant potential of PTH in the oncological field.

Does non-thermal plasma modify biopolymers in solution? A chemical and mechanistic study for alginate.

In the last decades, non-thermal plasma has been extensively investigated as a relevant tool for various biomedical applications, ranging from tissue decontamination to regeneration and from skin treatment to tumor therapies. This high versatility is due to the different kinds and amount of reactive oxygen and nitrogen species that can be generated during a plasma treatment and put in contact with the biological target. Some recent studies report that solutions of biopolymers with the ability to generate hydrogels, when treated with plasma, can enhance the generation of reactive species and influence their stability, resulting thus in the ideal media for indirect treatments of biological targets. The direct effects of the plasma treatment on the structure of biopolymers in water solution, as well as the chemical mechanisms responsible for the enhanced generation of RONS, are not yet fully understood. In this study, we aim at filling this gap by investigating, on the one hand, the nature and extent of the modifications induced by plasma treatment in alginate solutions, and, on the other hand, at using this information to explain the mechanisms responsible for the enhanced generation of reactive species as a consequence of the treatment. The approach we use is twofold: (i) investigating the effects of plasma treatment on alginate solutions, by size exclusion chromatography, rheology and scanning electron microscopy and (ii) study of a molecular model (glucuronate) sharing its chemical structure, by chromatography coupled with mass spectrometry and by molecular dynamics simulations. Our results point out the active role of the biopolymer chemistry during direct plasma treatment. Short-lived reactive species, such as OH radicals and O atoms, can modify the polymer structure, affecting its functional groups and causing partial fragmentation. Some of these chemical modifications, like the generation of organic peroxide, are likely responsible for the secondary generation of long-lived reactive species such as hydrogen peroxide and nitrite ions. This is relevant in view of using biocompatible hydrogels as vehicles for storage and delivery reactive species for targeted therapies.

Thermosensitive hydrogels to deliver reactive species generated by cold atmospheric plasma: a case study with methylcellulose.

Hydrogels have been recently proposed as suitable materials to generate reactive oxygen and nitrogen species (RONS) upon gas-plasma treatment, and postulated as promising alternatives to conventional cancer therapies. Acting as delivery vehicles that allow a controlled release of RONS to the diseased site, plasma-treated hydrogels can overcome some of the limitations presented by plasma-treated liquids in in vivo therapies. In this work, we optimized the composition of a methylcellulose (MC) hydrogel to confer it with the ability to form a gel at physiological temperatures while remaining in the liquid phase at room temperature to allow gas-plasma treatment with suitable formation of plasma-generated RONS. MC hydrogels demonstrated the capacity for generation, prolonged storage and release of RONS. This release induced cytotoxic effects on the osteosarcoma cancer cell line MG-63, reducing its cell viability in a dose-response manner. These promising results postulate plasma-treated thermosensitive hydrogels as good candidates to provide local anticancer therapies.

Chemical and Antimicrobial Effects of Air Non-Thermal Plasma Processing of Fresh Apple Juice with Focus on Safety Aspects.

Freshly squeezed apple juice was subjected to air non-thermal plasma treatment to investigate the capability of this processing method to inactivate microorganisms and to evaluate its safety when applied to liquid food products. Two different configurations of a transient spark discharge in ambient air were tested: an electrospray system with the juice flowing directly through the high voltage needle electrode, and a batch system, where the discharge was generated onto the surface of the juice. The key physico-chemical parameters of the juice, such as pH, conductivity, color, transmittance, and Brix degree, did not significantly change upon treatment. The concentration of nitrate ions formed by the plasma was safe, while that of nitrite ions and hydrogen peroxide was initially higher than the safety limits, but decreased within 24 h post treatment. The plasma effect on individual natural components of the juice, such as sugars, organic acids, and polyphenols, treated in water solutions led to their partial or substantial decomposition. However, when these compounds were plasma-treated altogether in the juice, they remained unaffected. The antimicrobial effect of the plasma processing was evaluated via the inoculation of model microorganisms. A stronger (6 log) decontamination was detected for bacteria Escherichia coli with respect to yeast Saccharomyces cerevisiae. Plasma processing led to a substantial extension of the juice shelf-life by up to 26 days if refrigerated, which represents a promising application potential in food technology.

Evaluation of the effects of cold atmospheric plasma and plasma-treated liquids in cancer cell cultures.

Cold atmospheric plasma (CAP) is a potential anticancer therapy. CAP has cytotoxic effects when applied either directly to cancer cell cultures or indirectly through plasma-conditioned liquids. This protocol describes how to treat adherent cultures of human cancer cell lines with CAP or plasma-conditioned medium and determine cell viability following treatment. The protocol also includes details on how to quantify the reactive oxygen and nitrogen species present in medium following CAP treatment, using chemical probes using UV-visible or fluorescence spectroscopy. CAP treatment takes ~30 min, and 3 h are required to complete quantification of reactive oxygen and nitrogen species. By providing a standardized protocol for evaluation of the effects of CAP and plasma-conditioned medium, we hope to facilitate the comparison and interpretation of results seen across different laboratories.

Quantification of Plasma-Produced Hydroxyl Radicals in Solution and their Dependence on the pH.

HO radicals are the most important reactive species generated during water treatment by non-thermal plasma devices. In this letter, we report the first quantification of the steady-state concentration and lifetime of plasma-produced hydroxyl radicals in water solutions at pH 3 and 7, and we discuss the differences based on their reactivity with other plasma-generated species. Finally, we show to what extent the use of chemical probes to quantify short-lived reactive species has an influence on the results and that it should be taken into account.

Enhanced Generation of Reactive Species by Cold Plasma in Gelatin Solutions for Selective Cancer Cell Death.

Atmospheric pressure plasma jets generate reactive oxygen and nitrogen species (RONS) in liquids and biological media, which find application in the new area of plasma medicine. These plasma-treated liquids were demonstrated recently to possess selective properties on killing cancer cells and attracted attention toward new plasma-based cancer therapies. These allow for local delivery by injection in the tumor but can be quickly washed away by body fluids. By confining these RONS in a suitable biocompatible delivery system, great perspectives can be opened in the design of novel biomaterials aimed for cancer therapies. Gelatin solutions are evaluated here to store RONS generated by atmospheric pressure plasma jets, and their release properties are evaluated. The concentration of RONS was studied in 2% gelatin as a function of different plasma parameters (treatment time, nozzle distance, and gas flow) with two different plasma jets. Much higher production of reactive species (H2O2 and NO2-) was revealed in the polymer solution than in water after plasma treatment. The amount of RONS generated in gelatin is greatly improved with respect to water, with concentrations of H2O2 and NO2- between 2 and 12 times higher for the longest plasma treatments. Plasma-treated gelatin exhibited the release of these RONS to a liquid media, which induced an effective killing of bone cancer cells. Indeed, in vitro studies on the sarcoma osteogenic (SaOS-2) cell line exposed to plasma-treated gelatin led to time-dependent increasing cytotoxicity with the longer plasma treatment time of gelatin. While the SaOS-2 cell viability decreased to 12%-23% after 72 h for cells exposed to 3 min of treated gelatin, the viability of healthy cells (hMSC) was preserved (∼90%), establishing the selectivity of the plasma-treated gelatin on cancer cells. This sets the basis for designing improved hydrogels with high capacity to deliver RONS locally to tumors.

Application of Fluorescence-Based Probes for the Determination of Superoxide in Water Treated with Air Non-thermal Plasma.

Superoxide is one of the reactive oxygen species (ROS) in non-thermal plasmas generated by electrical discharges in air at room temperature and atmospheric pressure. One important application of such plasmas is the activation of advanced oxidation processes for air and water decontaminating treatments. When in contact with aqueous media, ROS and notably superoxide can react at the plasma/liquid interface or transfer and react into the liquid. While the detection of superoxide in plasma-treated water has been reported in the literature, to the best of our knowledge, quantitative determinations are lacking. We report here the determination of superoxide rate of formation and steady-state concentration in water subjected to air non-thermal plasma in a streamer discharge reactor used previously to treat various organic contaminants. After detecting the presence of superoxide by spin-trapping and electron paramagnetic resonance analyses, we applied superoxide-selective fluorescent probes to carry out quantitative determinations. The first probe tested, 3',6'-bis(diphenylphosphinyl) fluorescein (PF-1), was not sufficiently soluble, but the second one, fluorescein-bis-[(N-methylpyridinium-3-yl)sulfonate iodide] (FMSI), was applied successfully. Under typical plasma operating conditions, the rate of superoxide formation and its steady-state concentration were (0.27 ± 0.15) µM s-1 and (0.007 ± 0.004) nM, respectively. The procedure outlined here can be usefully applied to detect and quantify superoxide in water treated by different plasma sources in various types of plasma reactors.

Production of reactive species in alginate hydrogels for cold atmospheric plasma-based therapies.

In the last years, great advances have been made in therapies based in cold atmospheric plasmas (CAP). CAP generate reactive oxygen and nitrogen species (RONS) which can be transferred to liquids. These CAP activated liquids display the same biological efficacy (i.e. on killing cancer cells) as CAP themselves, opening the door for minimally invasive therapies. However, injection of a liquid in the body results in fast diffusion due to extracellular fluids and blood flow. Therefore, the development of efficient vehicles which allow local confinement and delivery of RONS to the diseased site is a fundamental requirement. In this work, we investigate the generation of RONS (H2O2, NO2-, short-lived RONS) in alginate hydrogels by comparing two atmospheric pressure plasma jets: kINPen and a helium needle, at a range of plasma treatment conditions (time, gas flow, distance to the sample). The physic-chemical properties of the hydrogels remain unchanged by the plasma treatment, while the hydrogel shows several-fold larger capacity for generation of RONS than a typical isotonic saline solution. Part of the RONS are quickly released to a receptor media, so special attention has to be put on the design of hydrogels with in-situ crosslinking. Remarkably, the hydrogels show capacity for sustained release of the RONS. The plasma-treated hydrogels remain fully biocompatible (due the fact that the species generated by plasma are previously washed away), indicating that no cytotoxic modifications have occurred on the polymer. Moreover, the RONS generated in alginate solutions showed cytotoxic potential towards bone cancer cells. These results open the door for the use of hydrogel-based biomaterials in CAP-associated therapies.

Comment on "Water-Soluble Fluorescent Probe with Dual Mitochondria/Lysosome Targetability for Selective Superoxide Detection in Live Cells and in Zebrafish Embryos".

Recently, a new water-soluble, fluorescein-based probe for the detection of superoxide radical anion in aqueous media was developed by Lu et al. (ACS Sens. 2018, 3, 59-64). The probe was proven to be selective for superoxide and was used successfully also in cells and zebrafish embryos. To characterize the response of the probe to superoxide, Lu et al. used KO2 dissolved in deionized water as a surrogate. In testing this probe in different applications, we repeated some of these experiments and came to realize that the fluorescence signal observed by the Authors in their experiments with KO2 was incorrectly attributed to the reaction of the probe with superoxide and is due instead to its reactions with HO- and HO2-. We show that indeed under the conditions used in these assays KO2 undergoes very fast reaction with water to form HO- and HO2-. On the other hand, by using a proper surrogate, namely, KO2 dissolved in DMSO, and spin trapping experiments, we confirmed the ability of the probe to detect superoxide.

Radicals and Ions Formed in Plasma-Treated Organic Solvents: A Mechanistic Investigation to Rationalize the Enhancement of Electrospinnability of Polycaprolactone.

This paper reports and discusses the beneficial effects on the quality of electrospun polycaprolactone nanofibers brought about by pretreatment of the solvent with non-thermal plasma. Chloroform/dimethylformamide 9:1 (CHCl3:DMF 9:1) and pure chloroform were pretreated by a few minute exposure to the plasma generated by an atmospheric pressure plasma jet (APPJ). Interestingly, when pure chloroform was used, the advantages of plasma pretreatment of the solvent were way less pronounced than found with the CHCl3:DMF 9:1 mixture. The chemical modifications induced by the plasma in the solvents were investigated by means of complementary analytical techniques. GC-MS revealed the formation of solvent-derived volatile products, notably tetrachloroethylene (C2Cl4), 1,1,2,2-tetrachloroethane (C2H2Cl4), pentachloroethane (C2HCl5), hexachloroethane (C2Cl6) and, in the case of the mixed solvent, also N-methylformamide (C2H5NO). The chlorinated volatile products are attributed to reactions of ·Cl and Cl-containing methyl radicals and carbenes formed in the plasma-treated solvents. ·Cl and ·CCl3 radicals were detected and identified by EPR spectroscopy analyses. Ion chromatography revealed the presence of Cl-, NO 3 - , and HCOO- (the latter only in the presence of DMF) in the plasma-treated solvents, thus accounting for the observed increased conductivity and acidification of the solvent after plasma treatment. Mechanisms for the formation of these solvent derived products induced by plasma are proposed and discussed. The major role of radicals and ions in the plasma chemistry of chloroform and of the chloroform/dimethylformamide mixture is highlighted. The results provide insight into the interaction of plasma with organic solvents, a field so far little explored but holding promise for interesting applications.

Air non-thermal plasma treatment of Irgarol 1051 deposited on TiO2.

The herbicide 2-(methylthio)-4-(tert-butylamino)-6-(cyclopropylamino)-s-triazine (tradename Irgarol 1051, abbreviated here as Irg), widely used in antifouling paints as biocide inhibiting seaweeds growth, is found in coastal waters in the vicinity of ports and harbors. In this work, Irg was subjected to air non-thermal plasma (NTP) treatment, alone and in the presence of TiO2. A dielectric barrier discharge reactor was used, powered by AC voltage (18 kV, 50 Hz) to produce air-NTP directly above the surface of the aqueous Irg solution to be treated. Due to the very fast degradation of Irg occurring under the experimental conditions used, the results of kinetic experiments failed to detect any rate enhancement due to titania induced photodegradation. We show, however, that pre-adsorption of Irg on titania provides a means to significantly increase Irg NTP-induced degradation throughput, a result which might have useful practical consequences. It is concluded that this phenomenon is due to the acidic character of TiO2 which brings more Irg in solution by increasing the value of the ionization ratio, [IrgH+]/[Irg]. Product analysis, performed by LC/ESI-MSn, allowed us to detect and identify numerous intermediates of Irg degradation and to propose different competing reaction pathways for the investigated NTP induced Irg advanced oxidation process. The extent of mineralization to CO2 was assessed by Total Carbon analysis. It was found to reach 95% after 5 h treatment of Irg solutions with an initial concentration of 5·10-6 M. These results confirm the capability of our NTP prototype reactor to mineralize persistent organic pollutants.

Online Quantification of Criegee Intermediates of α-Pinene Ozonolysis by Stabilization with Spin Traps and Proton-Transfer Reaction Mass Spectrometry Detection.

Biogenic alkenes, which are among the most abundant volatile organic compounds in the atmosphere, are readily oxidized by ozone. Characterizing the reactivity and kinetics of the first-generation products of these reactions, carbonyl oxides (often named Criegee intermediates), is essential in defining the oxidation pathways of organic compounds in the atmosphere but is highly challenging due to the short lifetime of these zwitterions. Here, we report the development of a novel online method to quantify atmospherically relevant Criegee intermediates (CIs) in the gas phase by stabilization with spin traps and analysis with proton-transfer reaction mass spectrometry. Ozonolysis of α-pinene has been chosen as a proof-of-principle model system. To determine unambiguously the structure of the spin trap adducts with α-pinene CIs, the reaction was tested in solution, and reaction products were characterized with high-resolution mass spectrometry, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopy. DFT calculations show that addition of the Criegee intermediate to the DMPO spin trap, leading to the formation of a six-membered ring adduct, occurs through a very favorable pathway and that the product is significantly more stable than the reactants, supporting the experimental characterization. A flow tube set up has been used to generate spin trap adducts with α-pinene CIs in the gas phase. We demonstrate that spin trap adducts with α-pinene CIs also form in the gas phase and that they are stable enough to be detected with online mass spectrometry. This new technique offers for the first time a method to characterize highly reactive and atmospherically relevant radical intermediates in situ.

Local vs. external contribution to the budget of pollutants in the Po Valley (Italy) hot spot.

Numerical experiments using both passive tracers and full chemistry were performed to investigate the budget of pollutants in the Po Valley hot spot, using the BOLCHEM integrated model of atmospheric dynamics and composition. A one-year run was undertaken over the European domain, with a resolution of 50×50 km(2). Sources internal and external to the Po Valley were treated in separated runs. For the tracer experiment, the ratio between total masses of locally and externally emitted tracers showed that for half of the year the contribution from remote sources exceeds the local one. Although local sources prevailed at the ground level, external contribution exceedance was observed 15% of the time. Since the Po Valley is mostly surrounded by high mountains, vertical mixing and entrainment at the boundary layer top were found to be more effective than the advection at low levels. As the concentration of reacting species is affected by the nonlinearity of the transport-reaction mechanism, the results of the full chemistry runs with internal and external sources can be compared only in cases where this effect is weak. In these cases, the results for tracers were broadly confirmed for the less reacting species (CO, PM10) and, to some extent, also for more reactive ones (NO2, O3) for which more pronounced seasonal and diurnal cycles were found due to photochemical reactivity.

Numerical determination of personal aerosol sampler aspiration efficiency.

In this work the determination of the aspiration efficiency of personal aerosol samplers, commonly used in occupational exposure assessment, is investigated by means of CFD techniques. Specifically, it will be described a code to calculate the particle trajectories in a given flow field. At the present state the code considers only the effects of the mean flow field on the particle motion, whereas the turbulent diffusion effects are neglected. Comparisons with experimental measurements are also given in the framework of a research contract, supported by the European Community, with several experimental contributions from the participants. The main objective of the European research is to develop a new approach to experimentation with airborne particle flows, working on a reduced scale. This methodology has the advantage of allowing real-time aerosol determination and use of small wind tunnels, with a better experimental control. In this article we describe how the methodology has been verified using computational fluid dynamics. Experimental and numerical aspiration efficiencies have been compared and the influence of gravity and turbulence intensity in full and reduced scale has been investigated. The numerical techniques described here are in agreement with previous similar research and allow at least qualitative predictions of aspiration efficiency for real samplers, taking care of orientation from the incoming air flow. The major discrepancies among predicted and experimental results may be a consequence of bounce effects, which are very difficult to eliminate also by greasing the sampler surface.