Particulate and drug-induced toxicity assessed in novel quadruple cell human primary hepatic disease models of steatosis and pre-fibrotic NASH.

In an effort to replace, reduce and refine animal experimentation, there is an unmet need to advance current in vitro models that offer features with physiological relevance and enhanced predictivity of in vivo toxicological output. Hepatic toxicology is key following chemical, drug and nanomaterials (NMs) exposure, as the liver is vital in metabolic detoxification of chemicals as well as being a major site of xenobiotic accumulation (i.e., low solubility particulates). With the ever-increasing production of NMs, there is a necessity to evaluate the probability of consequential adverse effects, not only in health but also in clinically asymptomatic liver, as part of risk stratification strategies. In this study, two unique disease initiation and maintenance protocols were developed and utilised to mimic steatosis and pre-fibrotic NASH in scaffold-free 3D liver microtissues (MT) composed of primary human hepatocytes, hepatic stellate cells, Kupffer cells and sinusoidal endothelial cells. The characterized diseased MT were utilized for the toxicological assessment of a panel of xenobiotics. Highlights from the study included: 1. Clear experimental evidence for the pre-existing liver disease is important in the augmentation of xenobiotic-induced hepatotoxicity and 2. NMs are able to activate stellate cells. The data demonstrated that pre-existing disease is vital in the intensification of xenobiotic-induced liver damage. Therefore, it is imperative that all stages of the wide spectrum of liver disease are incorporated in risk assessment strategies. This is of significant consequence, as a substantial number of the general population suffer from sub-clinical liver injury without any apparent or diagnosed manifestations.

Class II resin composite restorations-tunnel vs. box-only in vitro and in vivo.

PURPOSE: In a combined in vitro/in vivo approach, tunnel vs. box-only resin composite restorations should be evaluated using thermomechanical loading (TML) in vitro and a restrospective clinical trial in vivo. MATERIALS AND METHODS: For the in vitro part, box-only and tunnel cavities were prepared in 32 extracted human third molars under simulated intraoral conditions in a phantom head. Specimens were randomly assigned to four groups (n = 8; 16 box-only/16 tunnel) and received bonded resin composite restorations with Amelogen Plus (box A/tunnel A) or lining with Ultraseal and Amelogen plus (box B/tunnel B) both bonded using PQ1 (all Ultradent). Specimens were subjected to a standardized aging protocol, 1-year water storage (WS) followed by TML (100,000 × 50 N; 2500 × + 5/+ 55 °C). Initially and after aging, marginal qualities were evaluated using replicas at × 200 magnification (SEM). For the corresponding in vivo observational study, 229 patients received 673 proximal resin composite restorations. From 371 tunnel restorations, 205 cavities were filled without flowable lining (tunnel A), and 166 tunnels were restored using UltraSeal as lining (tunnel B). A total of 302 teeth received conventional box-only fillings. Restorations were examined according to modified USPHS criteria during routine recalls up to 5 years of clinical service. RESULTS: In vitro, all initial results showed 100% gap-free margins when a flowable lining was used. Tunnels without lining exhibited some proximal shortcomings already before TML and even more pronounced after TML (p < 0.05). After TML, percentages of gap-free margins dropped to 87-90% in enamel with lining and 70-79% without lining (p < 0.05). In vivo, annual failure rates for box-only were 2.2%, for tunnel A 6.1%, and for tunnel B 1.8%, respectively (p < 0.05). Tunnels had significantly more sufficient proximal contact points than box-only restorations (p < 0.05). Flowable lining was highly beneficial for clinical outcome of tunnel-restorations (p < 0.05). CONCLUSIONS: With a flowable lining, tunnel restorations proved to be a good alternative to box-only resin composite restorations. CLINICAL RELEVANCE: Class II tunnel restorations showed to be a viable alternative for box-only restorations, however, only when flowable resin composite was used as adaptation promotor for areas being difficult to access.

Assessment of nanomaterial-induced hepatotoxicity using a 3D human primary multi-cellular microtissue exposed repeatedly over 21 days - the suitability of the in vitro system as an in vivo surrogate.

BACKGROUND: With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver's recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. RESULTS: The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. CONCLUSION: In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

Effects of Altering Magnesium Metal Surfaces on Degradation In Vitro and In Vivo during Peripheral Nerve Regeneration.

In vivo use of biodegradable magnesium (Mg) metal can be plagued by too rapid a degradation rate that removes metal support before physiological function is repaired. To advance the use of Mg biomedical implants, the degradation rate may need to be adjusted. We previously demonstrated that pure Mg filaments used in a nerve repair scaffold were compatible with regenerating peripheral nerve tissues, reduced inflammation, and improved axonal numbers across a short-but not long-gap in sciatic nerves in rats. To determine if the repair of longer gaps would be improved by a slower Mg degradation rate, we tested, in vitro and in vivo, the effects of Mg filament polishing followed by anodization using plasma electrolytic oxidation (PEO) with non-toxic electrolytes. Polishing removed oxidation products from the surface of as-received (unpolished) filaments, exposed more Mg on the surface, produced a smoother surface, slowed in vitro Mg degradation over four weeks after immersion in a physiological solution, and improved attachment of cultured epithelial cells. In vivo, treated Mg filaments were used to repair longer (15 mm) injury gaps in adult rat sciatic nerves after placement inside hollow poly (caprolactone) nerve conduits. The addition of single Mg or control titanium filaments was compared to empty conduits (negative control) and isografts (nerves from donor rats, positive control). After six weeks in vivo, live animal imaging with micro computed tomography (micro-CT) showed that Mg metal degradation rates were slowed by polishing vs. as-received Mg, but not by anodization, which introduced greater variability. After 14 weeks in vivo, functional return was seen only with isograft controls. However, within Mg filament groups, the amount of axonal growth across the injury site was improved with slower Mg degradation rates. Thus, anodization slowed degradation in vitro but not in vivo, and degradation rates do affect nerve regeneration.

In vitro percutaneous penetration test overview.

Skin is a detailed, organized, and intricate niche in the human body. Topical and transdermal drugs are unique, in that their absorption is quite different from other routes of administration (oral, intramuscular, intravenous, etc.,.). A robust amount of research is required to approve the use of a drug-in vivo, in vitro, and ex vivo studies collectively help manufacturers and government agencies with approval of various compounds. Use of human and animal studies poses ethical and financial concerns, making samples difficult to use. In vitro and ex vivo methods have improved over the past several decades-results show relevance when compared to in vivo methods. The history of testing is discussed, followed by a detailed account of known complexities of skin and the current state of percutaneous penetration.

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

In Vivo Effects of A Pro-PO System Inhibitor on the Phagocytosis of Xenorhabdus Nematophila in Galleria Mellonella Larvae.

Xenorhabdus nematophila is a Gram-negative bacterium symbiont of the entomopathogen nematode Steinernema carpocapsae whose immunosuppressive properties over host's immune response have been thoroughly investigated. In particular, live X. nematophila actively impairs phagocytosis in host's hemocytes through the secretion of inhibitors of eicosanoids synthesis. In this article we have investigated the cell surface structural features of X. nematophila responsible for the elusion from phagocytosis. To this end we have studied the uptake of heat-killed (hk), fluorescein isothiocyanate (FITC)-labeled X. nematophila by phagocytes from both a host insect and a mammalian species. In vitro dead X. nematophila passively resists engulfment by insect hemocytes without impairing the phagocytosis machinery whereas, unexpectedly, in vivo a significant phagocytosis of dead X. nematophila was observed. X. nematophila in vivo phagocytosis was increased by the co-injection of the specific inhibitor of pro-phenoloxidase (PO) system phenylthiourea (PTU), even if these effects were not observed in in vitro tests. Furthermore, biochemical modifications of X. nematophila cell wall implement in vivo phagocytosis, suggesting that this bacterium avoid phagocytosis because the ligand of phagocytic receptors is somehow buried or disguised in the cell wall. Finally, dead X. nematophila escapes engulfment even by human phagocytes suggesting that X. nematophila could be a useful model to investigate escape from phagocytosis by mammalian macrophages.

Specific Surface Modifications of Silica Nanoparticles Diminish Inflammasome Activation and In Vivo Expression of Selected Inflammatory Genes.

Silica (SiO2) nanoparticles (NPs) usage includes, but is not limited to, industrial and biomedical applications. Toxic effects of SiO2 NPs have been explored either in vitro or in vivo, assessing different surface modifications to reduce their harmful effects. Here, murine bone marrow-derived dendritic (BMDC) and a mouse model of mild allergic inflammation were used to study inflammasome activation and lung inflammation. Our results showed that SiO2 plain NPs induced NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome activation, increasing interleukin (IL)-1ß release in vitro, and, to a lesser extent, in vivo. In addition, SiO2 plain NPs triggered a pulmonary inflammatory milieu in both non-sensitized (NS) and sensitized (S) mice, by inducing the expression of key inflammatory cytokines and chemokines. Electron microscopy showed that SiO2 NPs were mostly localized in alveolar macrophages, within vesicles and/or in phagolysosomes. Both the in vitro and the in vivo effects of SiO NPs were attenuated by coating NPs with phosphonate or amino groups, whereas PEGylation, although it mitigated inflammasome activation in vitro, was not a successful coating strategy in vivo. These findings highlight that multiple assays are required to determine the effect of surface modifications in limiting NPs inflammatory potential. Taken together, these data are obtained by comparing in vitro and in vivo effects of SiO2 NPs suggest the use of amino and phosphonate coating of silica NPs for commercial purposes and targeted applications, as they significantly reduce their proinflammatory potential.