Search

Bone metastases occur in 65%–80% advanced breast cancer patients. Although significant progresses have been made in understanding the biological mechanisms driving the bone metastatic cascade, traditional 2D in vitro models and animal studies are not effectively reproducing breast cancer cells (CCs) interactions with the bone microenvironment and suffer from species-specific differences, respectively. Moreover, simplified in vitro models cannot realistically estimate drug anti-tumoral properties and side effects, hence leading to pre-clinical testing frequent failures. To solve this issue, a 3D metastatic bone minitissue (MBm) is designed with embedded human osteoblasts, osteoclasts, bone-resident macrophages, endothelial cells and breast CCs. This minitissue recapitulates key features of the bone metastatic niche, including the alteration of macrophage polarization and microvascular architecture, along with the induction of CC micrometastases and osteomimicry. The minitissue reflects breast CC organ-specific metastatization to bone compared to a muscle minitissue. Finally, two FDA approved drugs, doxorubicin and rapamycin, have been tested showing that the dose required to impair CC growth is significantly higher in the MBm compared to a simpler CC monoculture minitissue. The MBm allows the investigation of metastasis key biological features and represents a reliable tool to better predict drug effects on the metastatic bone microenvironment.

Bioactive glasses (BGs), due to their ability to influence osteogenic cell functions, have become attractive materials to improve loaded and unloaded bone regeneration. BG systems can be easily doped with several metallic ions (e.g., Ag, Sr, Cu, Nb) in order to confer antibacterial properties. In particular, Nb, when compared with other metal ions, has been reported to be less cytotoxic and possess the ability to enhance mineralization process in human osteoblast populations. In this study, we co-deposited, through one-pot electrophoretic deposition (EPD), chitosan (CS), gelatin (GE) and a modified BG containing Nb to obtain substrates with antibacterial activity for unloaded bone regeneration. Self-standing composite scaffolds, with a defined porosity (15–90 μm) and homogeneous dispersion of BGs were obtained. TGA analysis revealed a BG loading of about 10% in the obtained scaffolds. The apatite formation ability of the scaffolds was evaluated in vitro in simulated body fluid (SBF). SEM observations, XRD and FT-IR spectra showed a slow (21–28 days) yet effective nucleation of CaP species on BGs. In particular, FT-IR peak around 603 cm−1 and XRD peak at 2θ = 32°, denoted the formation of a mineral phase after SBF immersion. In vitro biological investigation revealed that the release of Nb from composite scaffolds had no cytotoxic effects. Interestingly, BG-doped Nb scaffolds displayed antibacterial properties, reducing S. lutea and E. coli growth of ≈60% and ≈50%, respectively. Altogether, the obtained results disclose the produced composite scaffolds as promising materials with inherent antibacterial activity for bone tissue engineering applications.

Current COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has put a spotlight on the spread of infectious diseases brought on by pathogenic airborne bacteria and viruses. In parallel with a relentless search for therapeutics and vaccines, considerable effort is being expended to develop ever more powerful technologies to restricting the spread of airborne microorganisms in indoor spaces through the minimization of health- and environment-related risks. In this context, UV-based and photocatalytic oxidation (PCO)-based technologies (i.e., the combined action of ultraviolet (UV) light and photocatalytic materials such as titanium dioxide (TiO2)) represent the most widely utilized approaches at present because they are cost-effective and ecofriendly. The virucidal and bactericidal effect relies on the synergy between the inherent ability of UV light to directly inactivate viral particles and bacteria through nucleic acid and protein damages, and the production of oxidative radicals generated through the irradiation of the TiO2 surface. In this literature survey, we draw attention to the most effective UV radiations and TiO2-based PCO technologies available and their underlying mechanisms of action on both bacteria and viral particles. Since the fine tuning of different parameters, namely the UV wavelength, the photocatalyst composition, and the UV dose (viz, the product of UV light intensity and the irradiation time), is required for the inactivation of microorganisms, we wrap up this review coming up with the most effective combination of them. Now more than ever, UV- and TiO2-based disinfection technologies may represent a valuable tool to mitigate the spread of airborne pathogens.

A thorough understanding of cell response to combined culture configuration and mechanical cues is of paramount importance in vascular tissue engineering applications. Herein, we investigated and compared the response of vascular smooth muscle cells (vSMCs) cultured in different culture environments (2D cell monolayers and 3D cellularized collagen-based gels) in combination with mechanical stimulation (7% uniaxial cyclic strain, 1 Hz) for 2 and 5 days. When cyclic strain was applied, two different responses, in terms of cell orientation and expression of contractile-phenotype proteins, were observed in 2D and 3D models. Specifically, in 2D configuration, cyclic strain caused ∼50% of cell population to align nearly perpendicular (80-90 degrees) to the strain direction, while not influencing the contractile-phenotype protein expression, as compared to the 2D static controls. Conversely, the application of uniaxial strain to 3D constructs induced a ∼60% cell alignment almost parallel (0-10 degrees) to the strain direction. Moreover, 3D mechanical stimulation applied for 5 days induced a twofold increase of SM α-actin level and a 14-fold increase of calponin expression as compared to 3D static controls. Altogether these findings provide a new insight into the potential to drive cell behavior by modulating the extracellular matrix and the biomechanical environment. Biotechnol. Bioeng. 2016;113: 2254-2263. © 2016 Wiley Periodicals, Inc.

This paper contains original data supporting the antibacterial activities of Gallium (Ga3+)-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in “The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii” (Cochis et al. 2016) [1].In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga3+ enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga3+ activity were compared to Silver. Keywords: E. coli, S. epidermidis, Biofilm, Gallium, Silver, Titanium, Anodic Spark Deposition

The decoration of polyamidoamine (PAMAM) dendrimers with some functional moieties holds promises for improving the gene-delivery behavior of the resulting polyplexes. Herein, we reported the synthesis and the development of non-viral gene vectors built on an array of PAMAMs, spanning generation 2 (G2), G4, and G7, specifically tethered with multivalent neomycin (Neo) or guanidinoneomycin (GNeo). Following a thorough chemical characterization by means of NMR and MALDI analyses, the ability of PAMAM–(G)Neo conjugates to serve as transfectants was investigated. First, we demonstrated that every PAMAM, whether or not tethered with Neo (or GNeo), exhibited greater DNA packing ability than the gold standard transfectant 25 kDa bPEI. Measurements of the colloidal properties showed that PAMAMs, when mixed with plasmid DNA (pDNA) as a function of the nitrogen-to-phosphate ratio (N/P), allowed preparing complexes of ≈150–300 nm in diameter with a positive surface charge of ≈+20–35 mV, strictly dependent on the PAMA...

BACKGROUND: We systematically analyzed the synergistic effect of: i) cytokine-mediated inflammatory activation of endothelial cells (ECs), and ii) shear-mediated platelet activation (SMPA) as a potential contributory mechanism to intraventricular thrombus formation in the setting of Left Ventricular Assist Device (LVAD) support. METHODS: Intact and shear-activated human platelets were exposed to nonactivated and cytokine-activated ECs. To modulate the level of LVAD-related shear activation, platelets were exposed to shear stress patterns of varying magnitude (30, 50, 70 dynes/cm(2), 10min) via a Hemodynamic Shearing Device. ECs were activated via exposure to inflammatory Tumor Necrosis Factor-α (TNF-α, 10 and 100 ng/mL, 24h), consistent with inflammatory activation recorded in patients on LVAD circulatory support. RESULTS: Adhesivity of shear-activated platelets to ECs was significantly higher than that of intact/unactivated platelets, regardless of the initial activation level (70 dynes/cm(2) shear-activated platelets vs. intact platelets: +80%, p

Background: Great interest has recently been focused on tooth and tooth derivatives as suitable substrates for the treatment of alveolar bone defects. Here, we propose the use of demineralized baby teeth (BT) as potential grafting materials for bone augmentation procedures. Methods: Particles of human BT (Ø < 1 mm) were demineralized by means of a chemical/thermal treatment. Demineralized BT particles were thoroughly characterized by scanning electron microscopy/energy dispersive X-ray analyses to evaluate the effects of the demineralization on BT topography and mineral phase composition, and by enzyme-linked immunosorbent assays (ELISA) to quantify collagen and bone morphogenetic protein-2 (BMP-2) protein contents. The response of SAOS-2 cells to exogenous BMP-2 stimulation was evaluated to identify the minimum BMP-2 concentration able to induce osteodifferentiation in vitro (alkaline phosphatase (ALP) activity). Results: The demineralization treatment led to a dramatic decrease in relative Ca and P content (%) of ≈75% with respect to the native BT particles, while preserving native protein conformation and activity. Interestingly, the demineralization process led to a rise in the bioavailability of BMP-2 in BT particles, as compared to the untreated counterparts. The BMP-2 content found in demineralized BT was also proved to be very effective in enhancing ALP activity, thus in the osteodifferentiation of SAOS-2 cells in vitro, as confirmed by cell experiments performed upon exogenously added BMP-2. Conclusions: In this study we demonstrate that the BMP-2 content found in demineralized BT is very effective in inducing cell osteodifferentiation, and strengthens the idea that BTs are very attractive bioactive materials for bone-grafting procedures.

The catalytic role of N-hydroxyphthalimide (NHPI) in promoting free-radical hydrogen atom transfer (HAT) reactions, well-documented for processes of industrial and synthetic interest, is here investigated for the first time in a biological environment. While NHPI by itself did not show any bioactivity, selected NHPI-derivatives (NHPIDs) revealed the ability to activate the intracellular formation of reactive oxygen species (ROS), causing the depletion of glutathione (GSH) levels and an increase in oxidative stress (OS). The evident bioactivity of some of these derivatives resulted in a significant reduction of the viability in osteosarcoma MG-63, suggesting a new, potential role of NHPIDs as pro-oxidant drugs. The key role of the N–OH group in promoting oxidative stress is demonstrated.

One of the tightest bottlenecks in vascular tissue engineering (vTE) is the lack of strength and elasticity of engineered vascular wall models caused by limited elastic fiber deposition. In this study, flat and tubular collagen gel-based scaffolds were cellularised with vascular smooth muscle cells (SMCs) and supplemented with human plasma fibronectin (FN), a known master organizer of several extracellular matrix (ECM) fiber systems. The consequences of FN on construct maturation was investigated in terms of geometrical contraction, viscoelastic mechanical properties and deposition of core elastic fiber proteins. FN was retained in the constructs and promoted deposition of elastin by SMCs as well as of several proteins required for elastogenesis such as fibrillin-1, lysyl oxidase, fibulin-4 and latent TGF-β binding protein-4. Notably, gel contraction, tensile equilibrium elastic modulus and elasticity were strongly improved in tubular engineered tissues, approaching the behaviour of native arteries. In conclusion, this study demonstrates that FN exerts pivotal roles in directing SMC-mediated remodeling of scaffolds toward the production of a physiological-like, elastin-containing ECM with excellent mechanical properties. The developed FN-supplemented systems are promising for tissue engineering applications where the generation of mature elastic tissue is desired and represent valuable advanced in vitro models to investigate elastogenesis.

Polyplexes (PX) are soft materials, obtained by blending polycations and nucleic acids, designed for gene delivery applications. While much is known about the transfection properties of PX, their protein corona, the biomolecules interacting with colloids once in a biological environment, represents an underlooked parameter in gene transfection. In this study, linear and branched polyethylenimines (lPEI and bPEI), the golden standard among non-viral vectors, were selected and used throughout the work: their physicochemical properties and protein corona when complexed to DNA were studied and linked to the toxicity and transfection efficiency arisen upon their delivery to cells. Interestingly, lPEIDNA and bPEIDNA complexes were characterized by similar physicochemical features, but different biological behavior. In fact, the biological milieu where cells and PX interact greatly influences their size, stability and transfection abilities. Using PX as a soft material model system, we spotlighted structure-activity relationships and methodologies that can help interpret their biological behavior and guide future studies in the field.

Zinc-doped hydroxyapatite (HA + Zn) has aroused increasing interest in bone contact applications, for its capacity to modulate osteoblast activity and for the antibacterial properties of zinc ions. In this study, HA + Zn coatings deposited by the Solution Precursor Plasma Spraying (SPPS) process were characterized. This method employs solution feedstocks providing simple and effective ion doping and resulting in the deposition of lamellae of smaller size than the conventional Atmospheric Plasma Spraying method. After a comprehensive microstructural characterization of the coatings, their bioactivity, cytotoxicity, and antibacterial efficacy were investigated. The coatings' bioactivity was confirmed by soaking them in Simulated Body Fluid (SBF), a commonly-used protocol to evaluate samples' bioactivity in vitro. Further, the coatings were non-cytotoxic against human osteoblast Saos-2-cells and, additionally, they showed antibacterial effects against Escherichia coli and Staphylococcus aureus. The HA + Zn coatings obtained showed higher efficacy against the gram-positive S. aureus than against the gram-negative Escherichia coli. These findings demonstrate the potential of the SPPS method for fabricating Zn-doped hydroxyapatite coatings, and show that Zn ions impart antibacterial properties against E. coli and S. aureus bacteria.

Background In recent decades, tooth derivatives such as dentin (D) and enamel (E) have been considered as potential graft biomaterials to treat bone defects. This study aimed to investigate the effects of demineralization on the physical-chemical and biological behavior of D and E. Methods Human D and E were minced into particles (ØResults The sterilization process, as a combination of chemical and thermal treatments, was found to be effective for all materials. On the other hand, D demineralization allowed preserving the collagen content, while increasing BMP-2 bioavailability. D and demineralized D (dD) displayed excellent biocompatibility, even greater than Bio-Oss®. Conversely, the high mineral content displayed by E, as confirmed by EDS analysis, inhibited cell proliferation. Of note, even though the demineralization process was somehow less effective in E than in D, demineralized E (dE) displayed increased BMP-2 bioavailability and improved performance in vitro compared with native E. Conclusions Our results substantiate the idea that the demineralization process lead to an increase of BMP-2 bioavailability, thus paving the way toward development of more effective, osteoinductive tooth-derived materials for bone regeneration and replacement.

The development of tubular engineered tissues is a challenging research area aiming to provide tissue substitutes but also in vitro models to test drugs, medical devices, and even to study physiological and pathological processes. In this work, the design, fabrication, and validation of an original cost-effective tubular multilayered-tissue culture system (TMCS) are reported. By exploiting cellularized collagen gel as scaffold, a simple moulding technique and an endothelialization step on a rotating system, TMCS allowed to easily prepare in 48 h, trilayered arterial wall models with finely organized cellular composition and to mature them for 2 weeks without any need of manipulation. Multilayered constructs incorporating different combinations of vascular cells are compared in terms of cell organization and viscoelastic mechanical properties demonstrating that cells always progressively aligned parallel to the longitudinal direction. Also, fibroblast compacted less the collagen matrix and appeared crucial in term of maturation/deposition of elastic extracellular matrix. Preliminary studies under shear stress stimulation upon connection with a flow bioreactor are successfully conducted without damaging the endothelial monolayer. Altogether, the TMCS herein developed, thanks to its versatility and multiple functionalities, holds great promise for vascular tissue engineering applications, but also for other tubular tissues such as trachea or oesophagus.

BACKGROUND The plasma membrane controls the selective internalization of (macro)molecules through different mechanisms, often relaying on specialized outward-facing carriers such as exofacial proteins thiols (EPTs). Although the interchange of critical thiols and disulphides between EPTs and exogenous cargoes is the first critical event, the identification of specific cell interactors remains to be thoroughly explored. Besides, it is likewise evident that only the relatively little suite of EPTs truly reactive can be considered theranostic targets. OBJECTIVE We were aimed at developing a stepwise procedure for the isolation and identification of a subset of EPTs, that we named chemically reactive EPS, which are potential theranostic targets. METHOD In the present study, EPTs that displayed permissive sulfhydryls on the surface of live cells in vitro underwent i) chemo-selective capture by means of thiolated superparamagnetic microbeads (isolation step), followed by ii) their prompt release via disulphide breakage through the addition of DTT reducing agent (elution step) and iii) analysis by means of SDS-PAGE and LCMS/ MS (identification step). RESULTS In total cell lysates, most of the proteins recovered were intracellular. Conversely, this methodology allowed a 2.6-fold enrichment in chemically reactive EPTs recovered and identified, corresponding up to 37% of the total cellular proteins. The key element of our approach was the reversible chemo-selective capture through disulphide linkages between chemically reactive EPTs and free thiols on microbeads. CONCLUSION We devised an enabling methodology to selectively pick up, recover and characterize chemically reactive EPTs.

Background.Titanium and its alloy represent the commonly used material in orthopedic joint prosthetics and dental implantology fields due to their excellent mechanical properties and biocompatibility. Among the different causes of orthopaedic and dental implants failure, the event of infections still represents the most serious and devastating complication that may involve biomaterial devices. Purpose. The aim of the present work is to develop an innovative osteointegrative and antibacterial biomimetic coating and to perform the chemical-physical and in vitro biological characterization of the coating using SAOS-2 cell line. Among the objects we intend to study the antibacterial properties of the coating against both Gram-positive and Gram-negative bacteria strains. The treatment is developed using the Anodic Spark Deposition (ASD) electrochemical technique. Methods. The proposed antibacterial ASD treatment was obtained in an electrochemical solution containing silicon, calcium, phosphorous, sodium and silver nanoparticles. Surface morphology was characterized using SEM and laser profilometry. A qualitative analysis of the chemical composition of the coating was assessed by EDS. The adhesion properties of the coating to the titanium bulk were performed with a 3 points bending test. SAOS-2 osteoblastic cell line spreading and morphology were investigated using SEM and Phalloidin staining after 24h, 48h and 72h of cell culture on the different coating, while the viability was assessed using Alamar Blue assay after 1, 4 and 7 day of cell culture. The bacterial adhesion and the antibacterial properties were investigated after 3h and 24h of incubation with Streptococcus mutans, Streptococcus epidermidis and Escherichia Coli bacterial strains. Results. The proposed anodization treatment was capable to provide a chemical and morphological modified titanium oxide layer, adherent and characterized by micropourous morphology resulted enriched by calcium, silicon, phosphorous and silver. The preliminary biological characterization showed an optimal SAOS-2 cell adhesion and proliferation. A strong antibacterial effect against both Gram-positive and Gram-negative strain has been shown. Conclusions. Considering the results obtained in the present study, we believe that the novel biomimetic antibacterial treatment represents a promising treatment capable to enhance the osteointegration and contemporaneously confer antibacterial properties to titanium for dental and orthopaedic fields.

Covalent attachment of a phenolic antioxidant analogue of α-tocopherol to graphite-coated magnetic cobalt nanoparticles (CoNPs) provided a novel magnetically responsive antioxidant capable of preventing the autoxidation of organic materials and showing a reduced toxicity toward human cells.

The bioisosterism of the trifluoromethyl group, namely its capacity to act as a replacement for groups with similar size or shape without substantially altering key biological properties such as binding affinity, remains a controversial issue.

This paper describes the use of airborne hyperspectral remote sensing for mapping asbestos roofs in an orographic complex area in Northern Italy, the Aosta Valley. Using training samples collected during field surveys, thematic classification was able to detect the majority of asbestos surfaces. Considering the total amount of asbestos areas validation showed a correct detection of about 80%, while considering the number of asbestos roofs correctly detected this value decreased to 43%. This difference pointed out a clear relationship between data spatial resolution and asbestos roofs area. The study served as a first approach to an extensive use of the remote sensing technology for asbestos mapping over large areas and the encouraging results will support Public Administrations for decision making strategies and policies for their removal.

Purpose Despite improvements in operative environment and surgical techniques, post-operative infections remain one of the most devastating complications in total joint replacement prostheses. Several efforts have been made to modify the surface of materials in order to prevent bacterial adhesion and colonization. Here, we show a one-pot electrochemical surface modification process for co-deposition of calcium phosphate and gentamicin, with the aim of triggering specific biological responses and imparting antibacterial properties on titanium alloy prostheses. Methods Gentamicin-loaded calcium phosphate coatings were deposited on Ti specimens via cathodic polarization in an electrochemical bath containing different amounts of the antibiotic salt (1–10 mg mL–1). Coatings were evaluated in terms of chemico-physical properties, via SEM/EDX, XRD, and ICP analysis, and antibacterial activity, via agar disc diffusion test on Staphylococcus aureus 8325–4 and Staphylococcus aureus SA113. Results An effective incorporation of gentamicin was achieved without any major effect on the morphology and structure. Morphology resulted in a typical plate-like brushite structure, confirmed by chemical composition and crystallographic structure. Gentamicin-loaded coatings showed an antibacterial efficacy on both staphlococcal strains, with a dose-dependent activity. Conclusions Electrochemical technology can be advantageously exploited in order to obtain coatings for bone-contact prostheses with tailored antibacterial properties.

Regenerative medicine is a critical frontier in biomedical and clinical research. The major progresses in the last few years were driven by a strong clinical need which could benefit from regenerative medicine outcomes for the treatment of a large number of conditions including birth defects, degenerative and neoplastic diseases, and traumatic injuries. Regenerative medicine applies the principles of engineering and life sciences to enhance the comprehension of the fundamental biological mechanisms underlying the structure-function relationships in physiologic and pathologic tissues and to accomplish alternative strategies for developing in vitro biological substitutes which are able to restore, maintain, or improve tissue, and organ function. This paper reviews selected approaches currently being investigated at Politecnico di Milano in the field of regenerative medicine. Specific tissue-oriented topics are divided in three sections according to each developmental stage: in vitro study, pre-clinical study, and clinical application. In vitro studies investigate the basic phenomena related to gene delivery, stem cell behavior, tissue regeneration, and to explore dynamic culture potentiality in different applications: cardiac and skeletal muscle, cartilage, hematopoietic system, peripheral nerve, and gene delivery. Specific fields of regenerative medicine, i.e., bone, blood vessels, and ligaments engineering have already reached the preclinical stage providing promising insights for further research towards clinical applications. The translation of the results obtained during in vitro and preclinical steps into clinical organ replacement is a very challenging issue, which can offer a valid alternative to fight morbidity, organ shortage, and ethical-social problems associated with allotransplantation as shown in the clinical case reported in this review.

Synthetic vectors for gene delivery offer a wide variety of functional derivatization, which can be exploited to increase targeting and transfection efficacy. In this field, redox-sensitive agents based on the thiol/disulfide (–SH/–SS–) equilibrium are a class of promising transfectants. Here the thiol group content in lipoplexes formed by a triazine-based sulfhydryl surfactant (SH14) and a plasmid (pGEFP-N1) was probed by Electron Spin Resonance (ESR) of appropriately tailored nitroxides. By modelling the time decay of ESR intensity, details on the process of lipoplex formation were obtained. It was found that the concentration of available –SH groups depended on the contact time between SH14 and DNA, suggesting that lipoplex formation entailed disulfide bridge formation among SH14 molecules. This finding represents the first experimental evidence that the –SH/–SS– equilibrium plays a role in lipoplex formation when DNA is complexed by sulfhydryl-based transfecting agents, which may have profound influence on their mechanism of action.

BackgroundA promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined.Methodology/principal findingsWith the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25:50:25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro.Conclusions/significanceThe presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors.

A new bioactive titanium surface treatment, labeled Ti-ASD, was developed using the electrochemical anodic spark deposition (ASD) technique and results in a thickened titanium oxide layer with higher levels of calcium and phosphorus typical of newly deposited mineral phase. This study was aimed at extending the knowledge on Ti-ASD treatment, by means of evaluation of the attachment, morphology, proliferation, metabolic activity, differentiation, and mineralization of osteoblast-like cells (SaOS-2) after growth on Ti-ASD treated titanium compared with nontreated titanium (Ti) and with chemically etched titanium (Ti-ETC). This novel type of titanium coating supported cell attachment, cell proliferation, and mineralization, revealing no cytotoxicity effects. The expression of differentiation markers on Ti-ASD treated titanium shows that genes related to the proliferation phase (Collagen type I, Coll I; Cbfa-1) were early expressed, whereas genes related to the mineralization phase (alkaline phosphatase, osteopontin, bone sialo protein) increased in a time-related way. Mineralization occurred on all analyzed surfaces, but on Ti-ASD the number of bone-like nodules and the amount of mineralized area was higher. In conclusion, Ti-ASD resulted to be a good surface for osteoblast attachment and proliferation, also promoting the maintenance of cell differentiation and matrix mineralization, a fundamental requirement for sustain the osseointegration and the clinical success of dental implants. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009

We have studied an in vitro engineered cartilage model, consisting of bovine articular chondrocytes seeded on micro-porous scaffolds and perfused with very low regimens of interstitial flow. Our previous findings suggested that synthesis of sulphated glycosaminoglycans (sGAG) was promoted in this model, if the level of shear generated on cells was maintained below 10 mPa (0.1 dyn/cm2). Constructs were stimulated with a median shear stress of 1.2 and 6.7 mPa using two independent culture chambers. Quantification of the applied stresses and of oxygen consumption rates was obtained from computational modelling. Experimentally, we set a time zero reference at 24 hours after cell seeding and total culture time at two weeks. The cell metabolic activity, measured by MTT, was significantly lower in all constructs at two weeks (-73% in static controls, -66% in the 1.2 mPa group and -60% in the 6.7 mPa group) vs. the time zero group, and significantly higher (+33%) in the 7 mPa group vs. static controls. The ratio between synthesis of collagen type II/type I, measured by Western Blot, was significantly higher in the 1.2 mPa constructs (+109% vs. the 6.7 mPa group, +120% vs. the time zero group and +286% vs. static controls). A trend of decreased alpha-actin expression was observed with increased ratio of type II to type I collagen, in all groups. These results reinforce the notion that, at early time points in culture, hydrodynamic shear below 10 mPa may promote formation of extra-cellular matrix specific to hyaline cartilage in chondrocyte-seeded constructs.

Deficiency in cellular thiol tripeptide glutathione (l-gamma glutamyl-cysteinyl-glycine) determines the severity of several chronic and inflammatory human diseases that may be relieved by oral treatment with the glutathione precursor N-acetylcysteine (NAC). Here, we showed that the left ventricle (LV) of human failing heart was depleted in total glutathione by 54%. Similarly, 2-month post-myocardial infarction (MI) rats, with established chronic heart failure (CHF), displayed deficiency in LV glutathione. One-month oral NAC treatment normalized LV glutathione, improved LV contractile function and lessened adverse LV remodelling in 3-month post-MI rats. Biochemical studies at two time-points of NAC treatment, 3 days and 1 month, showed that inhibition of the neutral sphingomyelinase (N-SMase), Bcl-2 depletion and caspase-3 activation, were key, early and lasting events associated with glutathione repletion. Attenuation of oxidative stress, downregulation of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and its TNF-R1 receptor were significant after 1-month NAC treatment. These data indicate that, besides glutathione deficiency, N-SMase activation is associated with post-MI CHF progression, and that blockade of N-SMase activation participates to post-infarction failing heart recovery achieved by NAC treatment. NAC treatment in post-MI rats is a way to disrupt the vicious sTNF-alpha/TNF-R1/N-SMase cycle.

Background— Studies in isolated cardiomyocytes showed that replenishment in cellular glutathione, achieved with the glutathione precursor N -acetylcysteine (NAC), abrogated deleterious effects of tumor necrosis factor-α (TNF-α). Methods and Results— We examined the ability of NAC to limit the progression of cardiac injury in the rat model of hypertension, induced by the nitric oxide synthase inhibitor N G -nitro- l -arginine methyl ester (L-NAME) (50 mg/kg per day SC) and high-salt diet (HS) (8% NaCl). Four-week HS/L-NAME administration induced hypertension (193±8 versus 122±4 mm Hg for low-salt diet [LS] group) and left ventricular (LV) dysfunction, revealed by echocardiography and characterized by decreased LV shortening fraction (38±2% versus 49±4% for LS group; P P Conclusions— These findings suggest that glutathione status determines the adverse effects of TNF-α in cardiac failure and that TNF-α antagonism may be achieved by glutathione supplementation.

Purpose Despite widespread use of 3-dimensional (3D) micro-porous scaffolds to promote their potential application in cartilage tissue engineering, only a few studies have examined the response to hydrostatic pressure of engineered constructs. A high cyclic pressurization, currently believed to be the predominant mechanical signal perceived by cells in articular cartilage, was used here to stimulate bovine articular chondrocytes cultured in a synthetic 3D porous scaffold (DegraPol). Methods Construct cultivation lasted 3 days with applied pressurization cycles of amplitude 10 MPa, frequency 0.33 Hz, and stimulation sessions of 4 hours/day. Results At 3 days of culture, with respect to pre-culture conditions, the viability of the pressurized constructs did not vary, whereas it underwent a 16% drop in the unpressurized controls. Synthesis of α-actin was 34% lower in all cultured constructs. Synthesis of collagen II/collagen I did not vary in pressurized constructs, was 76% lower in unpressurized controls, and was around 230% higher in pressurized constructs with respect to unpressurized controls. Chondrocytes showed a phenotypic spherical morphology at time zero and at 3 days of pressurized culture. Conclusions Although the passage from 2D expansion to 3D geometry was effective to guide cell differentiation, only mechanical conditioning enabled the maintenance and further cell differentiation toward a mature chondrocytic phenotype.