Isolation, Ex Vivo Expansion, and Lentiviral Transduction of Alveolar Macrophages.

Alveolar macrophages (AM) are resident macrophages of the lung and play important roles in the maintenance of tissue homeostasis as well as host defense. Here, we describe how they can be harvested from murine lungs, expanded in vitro, and transduced with lentiviral vectors.

Local Preirradiation of Infarcted Cardiac Tissue Substantially Enhances Cell Engraftment.

The success of cell therapy for the treatment of myocardial infarction depends on finding novel approaches that can substantially implement the engraftment of the transplanted cells. In order to enhance cell engraftment, most studies have focused on the pretreatment of transplantable cells. Here we have considered an alternative approach that involves the preconditioning of infarcted heart tissue to reduce endogenous cell activity and thus provide an advantage to our exogenous cells. This treatment is routinely used in other tissues such as bone marrow and skeletal muscle to improve cell engraftment, but it has never been taken in cardiac tissue. To avoid long-term cardiotoxicity induced by full heart irradiation we developed a rat model of a catheter-based heart irradiation system to locally impact a delimited region of the infarcted cardiac tissue. As proof of concept, we transferred ZsGreen+ iPSCs in the infarcted heart, due to their ease of use and detection. We found a very significant increase in cell engraftment in preirradiated rats. In this study, we demonstrate for the first time that preconditioning the infarcted cardiac tissue with local irradiation can substantially enhance cell engraftment.

High glucose alters the secretome of mechanically stimulated osteocyte-like cells affecting osteoclast precursor recruitment and differentiation.

Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and ß-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1ß MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication.

Generation of iPSC from cardiac and tail-tip fibroblasts derived from a second heart field reporter mouse.

Mef2c Anterior Heart Field (AHF) enhancer is activated during embryonic heart development and it is expressed in multipotent cardiovascular progenitors (CVP) giving rise to endothelial and myocardial components of the outflow tract, right ventricle and ventricular septum. Here we have generated iPSC from transgenic Mef2c-AHF-Cre x Ai6(RCLZsGreen) mice. These iPSC will provide a novel tool to investigate the AHF-CVP and their cell progeny.

Effects of bleaching on osteoclast activity and their modulation by osteostatin and fibroblast growth factor 2.

HYPOTHESIS: Dental bleaching with H2O2 is a common daily practice in dentistry to correct discoloration of anterior teeth. The aim of this study has been to determine whether this treatment of human teeth affects growth, differentiation and activity of osteoclast-like cells, as well as the putative modulatory action of osteostatin and fibroblast growth factor 2 (FGF-2). EXPERIMENTS: Previously to the in vitro assays, structural, physical-chemical and morphological features of teeth after bleaching were studied. Osteoclast-like cells were cultured on human dentin disks, pre-treated or not with 38% H2O2 bleaching gel, in the presence or absence of osteostatin (100 nM) or FGF-2 (1 ng/ml). Cell proliferation and viability, intracellular content of reactive oxygen species (ROS), pro-inflammatory cytokine (IL-6 and TNFα) secretion and resorption activity were evaluated. FINDINGS: Bleaching treatment failed to affect either the structural or the chemical features of both enamel and dentin, except for slight morphological changes, increased porosity in the most superficial parts (enamel), and a moderate increase in the wettability degree. In this scenario, bleaching produced an increased osteoclast-like cell proliferation but decreased cell viability and cytokine secretion, while it augmented resorption activity on dentin. The presence of either osteostatin or FGF-2 reduced the osteoclast-like cell proliferation induced by bleaching. FGF-2 enhanced ROS content, whereas osteostatin decreased ROS but increased TNFα secretion. The bleaching effect on resorption activity was increased by osteostatin, but this effect was less evident with FGF-2. CONCLUSIONS: These findings further confirm the deleterious effects of tooth bleaching by affecting osteoclast growth and function as well as different modulatory actions of osteostatin and FGF-2.

Influence of the covalent immobilization of graphene oxide in poly(vinyl alcohol) on human osteoblast response.

The differences in the response of human Saos-2 osteoblasts to nanocomposites of poly(vinyl alcohol) (PVA) and 1.5wt.% graphene oxide (GO) prepared by covalent linking (PVA/GO-c) and simple blending (PVA/GO-m) have been evaluated through different biocompatibility parameters. The effects produced on osteoblasts by these two nanocomposites were analysed in parallel and compared with the direct action of GO and with the effect of PVA films without GO. The intracellular content of reactive oxygen species (ROS) and the levels of interleukin-6 (IL-6) were measured to evaluate oxidative stress induction and protective response, respectively. The results demonstrate that the combination of GO with PVA reduces both the proliferation delay and the internal cell complexity alterations induced by GO on human osteoblasts. Moreover, the covalent attachment of GO to the PVA chains increases both cell viability and IL-6 levels, reducing both apoptosis and intracellular ROS content when compared to simple blending of both materials. The use of this strategy to modulate the biointerface reduces the toxic effects of graphene while preserving the reinforcement characteristics for application in tissue engineering scaffolds, and has enormous interest for polymer/graphene biomaterials development.

Developmental effects and genotoxicity of 10 water disinfection by-products in zebrafish.

Disinfection by-products are contaminants produced during drinking water disinfection. Several DBPs have been implicated in a variety of toxic effects, mainly carcinogenic and genotoxic effects. Moreover, DBPs exposure has also been associated with an increased risk of developmental effects. In this study, the developmental toxicity and genotoxicity of 10 DBPs (four trihalomethanes [THMs], five haloacetic acids [HAAs] and sodium bromate) in the zebrafish embryo model were evaluated. Embryos exposed for 72 hours were observed for different endpoints such as growth, hatching success, malformations and lethality. THMs exposure resulted in adverse developmental effects and a significant reduced tail length. Two HAAs, tribromoacetic acid and dichloroacetic acid, along with sodium bromate were found to cause a significant increase in malformation rate. Chloroform, chlorodibromomethane and sodium bromate produced a weak induction of DNA damage to whole embryos. However, developmental effects occurred at a range of concentrations (20-100 µg/mL) several orders of magnitude above the levels that can be attained in fetal blood in humans exposed to chlorinated water. In conclusion, the teratogenic and genotoxic activity observed by some DBPs in zebrafish reinforce the view that there is a weak capacity of disinfection products to cause developmental effects at environmentally relevant concentrations.

Endocytic mechanisms of graphene oxide nanosheets in osteoblasts, hepatocytes and macrophages.

Nano-graphene oxide (GO) has attracted great interest in nanomedicine due to its own intrinsic properties and its possible biomedical applications such as drug delivery, tissue engineering and hyperthermia cancer therapy. However, the toxicity of GO nanosheets is not yet well-known and it is necessary to understand its entry mechanisms into mammalian cells in order to avoid cell damage and human toxicity. In the present study, the cellular uptake of pegylated GO nanosheets of ca. 100 nm labeled with fluorescein isothiocyanate (FITC-PEG-GOs) has been evaluated in the presence of eight inhibitors (colchicine, wortmannin, amiloride, cytochalasin B, cytochalasin D, genistein, phenylarsine oxide and chlorpromazine) that specifically affect different endocytosis mechanisms. Three cell types were chosen for this study: human Saos-2 osteoblasts, human HepG2 hepatocytes and murine RAW-264.7 macrophages. The results show that different mechanisms take part in FITC-PEG-GOs uptake, depending on the characteristics of each cell type. However, macropinocytosis seems to be a general internalization process in the three cell lines analyzed. Besides macropinocytosis, FITC-PEG-GOs can enter through pathways dependent on microtubules in Saos-2 osteoblasts, and through clathrin-dependent mechanisms in HepG2 hepatocytes and RAW-264.7 macrophages. HepG2 cells can also phagocytize FITC-PEG-GOs. These findings help to understand the interactions at the interface of GO nanosheets and mammalian cells and must be considered in further studies focused on their use for biomedical applications.

The effects of graphene oxide nanosheets localized on F-actin filaments on cell-cycle alterations.

Graphene oxide (GO) is considered to be a promising nanomaterial for biomedical applications due to its small two-dimensional shape besides its electrical and mechanical properties. However, only a few data concerning the cell responses to this material have been described and the GO biocompatibility has not been yet fully assessed. In the present study, graphene oxide nanosheets (GOs) decorated with 1-arm (1-GOs) and 6-arm (6-GOs) poly(ethylene glycol-amine) (PEG) have been incubated with cultured Saos-2 osteoblasts, MC3T3-E1 preosteoblasts and RAW-264.7 macrophages to analyze several key cell markers for in vitro biocompatibility evaluation. The results demonstrate that, after internalization, GO nanosheets are localized on F-actin filaments inducing cell-cycle alterations, apoptosis and oxidative stress in these cell types. The observed GOs effects must be considered in further studies focused on photothermal cancer therapy as a synergistic factor.

Síndrome de Gorlin (síndrome de carcinoma basocelular nevoide). Presentación de dos casos y revisión de la literatura / Gorlin's symdrome (nevoid basal cell carcinoma syndrome). Presentation of two cases and review of the literature

El síndrome de carcinoma basocelular nevoide fue descrito hace cuatro décadas por Gorlin y Goltz; ha sido caracterizado por múltiples carcinoma basocelulares en áreas expuestas o no al sol. Además, los pacientes con este síndrome cursan con quistes mandibulares, alteraciones esqueléticas (costilla bífida), puntilleo palmoplantar y calcificaciones ectópicas. Este síndrome está asociado con otras alteraciones de cara, de piel y de los sistemas musculoesquelético, genitourinario, neurológico y oftalmológico, así como algunas neoplasias, incluyendo, linfoma de Hodgkin, no-Hodgkin, meduloblastoma, fibromas de ovario y fibrosarcomas, melanoma y rabdomioma fetal. Se ha establecido que para el diagnóstico de síndrome de Gorlin, se requiere de dos características principales, consideradas como criterios mayores, o bien de una alteración mayor y dos menores. Algunos estudios han demostrado que este síndrome es una alteración autosómica dominante en el cromosoma 9, lo cual es relevante como un evento temprano en la carcinogénesis. Esta serie presenta dos casos de síndrome basocelular nevoide. En uno de ellos se efectuó estudio completo familiar y se detecto que había ocho miembros más de la familia que padecían esta enfermedad