The nano-revolution in the diagnosis and treatment of endometriosis.

Endometriosis is a painful gynecological disease with a high prevalence, affecting millions of women worldwide. Innovative, non-invasive treatments, and new patient follow-up strategies are needed to deal with the harmful social and economic effects. In this scenario, considering the recent, very promising results already reported in the literature, a commitment to new research in the field of nanomedicine is urgently needed. Study findings clearly show the potential of this approach in both the diagnostic and therapeutic phases of endometriosis. Here, we offer a brief review of the recent exciting and effective applications of nanomedicine in both the diagnosis and therapy of endometriosis. Special emphasis will be placed on the emerging theranostic application of nanoproducts, and the combination of phototherapy and nanotechnology as new therapeutic modalities for endometriosis. The review will also provide interested readers with a guide to the selection process and parameters to consider when designing research into this type of approach.

Internally crosslinked alginate-based bioinks for the fabrication ofin vitrohepatic tissue models.

Bioprinting is a key technique to fabricate cell-laden volumetric constructs with controlled geometry. It can be used not only to replicate the architecture of a target organ but also to produce shapes that allow for the mimicry,in vitro,of specific desired features. Among the various materials suitable to be processed with this technique, sodium alginate is currently considered one of the most appealing because of its versatility. To date, the most widespread strategies to print alginate-based bioinks exploit external gelation as a primary process, by directly extruding the hydrogel-precursor solution into a crosslinking bath or within a sacrificial crosslinking hydrogel, where the gelation takes place. In this work, we describe the print optimization and the processing of Hep3Gel: an internally crosslinked alginate and ECM-based bioink for the production of volumetric hepatic tissue models. We adopted an unconventional strategy, by moving from the reproduction of the geometry and the architecture of liver tissue to the use of bioprinting to fabricate structures that can promote a high degree of oxygenation, as is the case with hepatic tissue. To this end, the design of structures was optimized by employing computational methods. The printability of the bioink was then studied and optimized through a combination of differenta priorianda posteriorianalyses. We produced 14-layered constructs, thus highlighting the possibility to exploit internal gelation alone to directly print self-standing structures with finely controlled viscoelastic properties. Constructs loaded with HepG2 cells were successfully printed and cultured in static conditions for up to 12 d, underlining the suitability of Hep3Gel to support mid/long-term cultures.

Disassembling the complexity of mucus barriers to develop a fast screening tool for early drug discovery.

Mucus is a natural barrier with a protective role that hinders drug diffusion, representing a steric and interactive barrier to overcome for an effective drug delivery to target sites. In diseases like cystic fibrosis (CF), pulmonary mucus exhibits altered features, which hamper clearance mechanisms and drug diffusion, ultimately leading to lung failure. Effectively modelling the passage through mucus still represents an unmet challenge. An airway CF mucus model is herein proposed to disassemble the complexity of the mucus barrier following a modular approach. A hydrogel, mainly composed of mucin in an alginate (Alg) network, is proposed to specifically model the chemical-physical properties of CF mucus. The steric retention of pathological mucus was reproduced by targeting its mesh size (approximately 50 nm) and viscoelastic properties. The interactive barrier was reproduced by a composition inspired from the CF mucus. Optimized mucus models, composed of 3 mg ml-1 Alg and 25 mg ml-1 mucin, exhibited a G' increasing from ∼21.2 to 55.2 Pa and a G'' ranging from ∼5.26 to 28.8 Pa in the frequency range of 0.1 to 20 Hz. Drug diffusion was tested using three model drugs. The proposed mucus model was able to discriminate between the mucin-drug interaction and the steric barrier of a mucus layer with respect to the parallel artificial membrane permeability (PAMPA) that models the phospholipidic cell membrane, the state-of-the-art screening tool for passive drug diffusion. The mucus model can be proposed as an in vitro tool for early drug discovery, representing a step forward to model the mucus layer. Additionally, the proposed methodology allows to easily include other molecules present within mucus, as relevant proteins, lipids and DNA.

Polysaccharide-based hydrogels with tunable composition as 3D cell culture systems.

BACKGROUND: To date, cell cultures have been created either on 2-dimensional (2D) polystyrene surfaces or in 3-dimensional (3D) systems, which do not offer a controlled chemical composition, and which lack the soft environment encountered in vivo and the chemical stimuli that promote cell proliferation and allow complex cellular behavior. In this study, pectin-based hydrogels were developed and are proposed as versatile cell culture systems. METHODS: Pectin-based hydrogels were produced by internally crosslinking pectin with calcium carbonate at different initial pH, aiming to control crosslinking kinetics and degree. Additionally, glucose and glutamine were added as additives, and their effects on the viscoelastic properties of the hydrogels and on cell viability were investigated. RESULTS: Pectin hydrogels showed in high cell viability and shear-thinning behavior. Independently of hydrogel composition, an initial swelling was observed, followed by a low percentage of weight variation and a steady-state stage. The addition of glucose and glutamine to pectin-based hydrogels rendered higher cell viability up to 90%-98% after 1 hour of incubation, and these hydrogels were maintained for up to 7 days of culture, yet no effect on viscoelastic properties was detected. CONCLUSIONS: Pectin-based hydrogels that offer tunable composition were developed successfully. They are envisioned as synthetic extracellular matrix (ECM) either to study complex cellular behaviors or to be applied as tissue engineering substitutes.

Synthesis and characterization of strontium-substituted hydroxyapatite nanoparticles for bone regeneration.

The production of stable suspensions of strontium-substituted hydroxyapatite (Sr-HA) nanopowders, as Sr ions vector for bone tissue regeneration, was carried out in the present work. Sr-HA nanopowders were synthesized via aqueous precipitation methods using Sr2+ amount from 0 to 100mol% and were characterized by several complementary techniques such as solid-state Nuclear Magnetic Resonance spectroscopy, X-ray diffraction, Infrared spectroscopy, N2 physisorption and Transmission Electron Microscopy. The substitution of Ca2+ with Sr2+ in HA is always isomorphic with gradual evolution between the two limit compositions (containing 100% Ca and 100% Sr), this pointing out the homogeneity of the synthesized nanopowders and the complete solubility of strontium in HA lattice. Strontium addition is responsible for an increasing c/a ratio in the triclinic unit cell. A significant variation of the nanopowders shape and dimension is also observed, a preferential growth along the c-axis direction being evident at higher strontium loads. Modifications in the local chemical environment of phosphate and hydroxyl groups in the apatite lattice are also observed. Stable suspensions were produced by dispersing the synthesized nanopowders in bovine serum albumin. Characterization by Dynamic Light Scattering and ζ-potential determination allowed to show that Ca2+→Sr2+ substitution influences the hydrodynamic diameter, which is always twice the particles size determined by TEM, the nanoparticles being always negatively charged as a result from the albumin rearrangement upon the interaction with nanoparticles surface. The biocompatibility of the suspensions was studied in terms of cell viability, apoptosis, proliferation and morphology, using osteosarcoma cell line SAOS-2. The data pointed out an increased cell proliferation for HA nanoparticles containing larger Sr2+ load, the cells morphology remaining essentially unaffected.

Reactive hydroxyapatite fillers for pectin biocomposites.

In this work, a novel injectable biocomposite hydrogel is produced by internal gelation, using pectin as organic matrix and hydroxyapatite either as crosslinking agent and inorganic reinforcement. Tunable gelling kinetics and rheological properties are obtained varying the hydrogels' composition, with the final aim of developing systems for cell immobilization. The reversibility by dissolution of pectin-hydroxyapatite hydrogels is achieved with saline solutions, to possibly accelerate the release of the cells or active agents immobilized. Texture analysis confirms the possibility of extruding the biocomposites from needles with diameters from 20 G to 30 G, indicating that they can be implanted with minimally-invasive approaches, minimizing the pain during injection and the side effects of the open surgery. L929 fibroblasts entrapped in the hydrogels survive to the immobilization procedure and exhibit high cell viability. On the overall, these systems result to be suitable supports for the immobilization of cells for tissue regeneration applications.

Injectable pectin hydrogels produced by internal gelation: pH dependence of gelling and rheological properties.

The production of injectable pectin hydrogels by internal gelation with calcium carbonate is proposed. The pH of pectin was increased with NaOH or NaHCO3 to reach physiological values. The determination of the equivalence point provided evidence that the pH can be more precisely modulated with NaHCO3 than with NaOH. Degradation and inability to gel was observed for pectin solutions with pH 5.35 or higher. Therefore, pectin solutions with pH values varying from 3.2 (native pH) to 3.8 were chosen to produce the gels. The increase of the pH for the crosslinked hydrogels, as well as the reduction of the gelling time and their thickening, was dependent upon the amount of calcium carbonate, as confirmed by rheology. Hydrogel extracts were not cytotoxic for L-929 fibroblasts. On the overall, the investigated formulations represent interesting injectable systems providing an adequate microenvironment for cell, drug or bioactive molecules delivery.

Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors.

Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm; 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm(2). The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

Environmentally friendly lycopene purification from tomato peel waste: enzymatic assisted aqueous extraction.

The antioxidant and anticancer properties of lycopene make it an ideal component for daily food supplements. For this reason this study investigated the possibility of extracting lycopene from tomato waste peels using a green chemistry protocol devoid of organic solvent. Cells are lysed thanks to a combination of pH changes and hydrolytic enzyme treatments. The lycopene-containing chromoplasts are then precipitated by lowering the pH and isolated through a centrifugation step. At this stage the lycopene content of the isolated chromoplasts shows a 10-fold increase (3-5% w/w, dry basis) with respect to untreated tomato peels. A further improvement in lycopene concentration is obtained by a second enzymatic treatment using a protease cocktail. This catalytic step eliminates unwanted proteins, bound to the chromoplasts, but not essential for their stability. The final product shows a lycopene content around 8-10% (w/w, dry basis), which represents a 30-fold increase with respect to the lycopene concentration of the untreated peels.

SEM evaluation of the root canal walls after treatment with Tetraclean.

PURPOSE: In this ex vivo study, the surface cleanliness of root canal walls after shaping with Ni-Ti instruments and irrigating with different endodontic solutions was evaluated by SEM. METHODS: 50 single-rooted teeth were divided into 5 groups. All root canals were shaped with Revo-S™ (MICRO-MEGA®, Besancon, France) rotary Ni-Ti instruments and irrigated with NaOCl, varying the final rinse: NaOCl, Tetraclean, 17% EDTA, Cloreximid, Tetraclean liquid not mixed with powder. Specimens were fractured longitudinally and prepared for SEM analysis in order to evaluate the presence/absence of smear layer and the presence/absence of open tubules at the coronal, middle, and apical third of each canal, using a 3-step scale for scores. Numeric data were analyzed using Kruskall-Wallis test and Mann-Whitney U test and significance was predetermined at p<0.05. RESULTS: This study showed significant differences between the various groups. The 5.25% NaOCl + Tetraclean and 5.25% NaOCl + 17% E.D.T.A. groups had significantly lower scores than other groups (p<0.001), with no significant difference between them. CONCLUSIONS: Final rinse with Tetraclean is effective in removing the smear layer, leaving a high number of opened dentin tubules.

The effect of photodynamic treatment combined with antibiotic action or host defence mechanisms on Staphylococcus aureus biofilms.

Staphylococcus aureus is one of the most important etiological agents of infections associated with medical devices. This is in part due to the ability of the organism to form biofilm, which provides a microenvironment that protects from attack by the host's immune system and by antibiotics. In this study we examined the structure of polysaccharide intercellular adhesin (PIA)-dependent or protein-based S. aureus biofilms. We defined new strategies aimed at treatment of mature established biofilms using photodynamic treatment (PDT) combined with chemotherapy or phagocytosis. Significant inactivation of bacteria was observed when structurally distinct biofilms were exposed to the cationic porphyrin, tetra-substituted N-methyl-pyridyl-porphine (TMP), and simultaneously to visible light. Moreover, PDT-treated biofilms exposed to vancomycin or subjected to the phagocytic action of whole blood resulted in their almost complete eradication. The drastic reduction in staphylococcal survival and the disruption of biofilms were confirmed by confocal laser scanning microscopy and scanning electron microscopy. The results suggest that PDT combined with vancomycin and the host defences may be a useful approach for the inactivation of staphylococcal biofilms adhering to medical implant surfaces.

Enhanced in vitro culture of human SAOS-2 osteoblasts on a sand-blasted titanium surface modified with plastic deformation.

The titanium surfaces with micro-roughness have been studied to substitute machined titanium, with the focus on enhancing the bone apposition onto the implant. In this study we have followed a biomimetic strategy where human SAOS-2 osteoblasts proliferated and built their extracellular matrix on a sandblasted titanium surface modified with plastic deformation. In comparison with sandblasted titanium surface, the plastic deformation increased the cell proliferation and the surface coating with bone matrix. The superficially modified biomaterial could be used, in clinical applications, as an implant for bone repair.