Differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of Mannheimia haemolytica.

Mannheimia haemolytica is the primary bacterial species associated with respiratory disease of ruminants. A lack of cost-effective, reproducible models for the study of M. haemolytica pathogenesis has hampered efforts to better understand the molecular interactions governing disease progression. We employed a highly optimised ovine tracheal epithelial cell model to assess the colonisation of various pathogenic and non-pathogenic M. haemolytica isolates of bovine and ovine origin. Comparison of single representative pathogenic and non-pathogenic ovine isolates over ten time-points by enumeration of tissue-associated bacteria, histology, immunofluorescence microscopy and scanning electron microscopy revealed temporal differences in adhesion, proliferation, bacterial cell physiology and host cell responses. Comparison of eight isolates of bovine and ovine origin at three key time-points (2 h, 48 h and 72 h), revealed that colonisation was not strictly pathogen or serotype specific, with isolates of serotype A1, A2, A6 and A12 being capable of colonising the cell layer regardless of host species or disease status of the host. A trend towards increased proliferative capacity by pathogenic ovine isolates was observed. These results indicate that the host-specific nature of M. haemolytica infection may result at least partially from the colonisation-related processes of adhesion, invasion and proliferation at the epithelial interface.

Tri-modal imaging of gold-dotted magnetic nanoparticles for magnetic resonance imaging, computed tomography and intravascular ultrasound: an in vitro study.

Aim: To examine the multimodal contrasting ability of gold-dotted magnetic nanoparticles (Au*MNPs) for magnetic resonance (MR), computed tomography (CT) and intravascular ultrasound (IVUS) imaging. Materials & methods: Au*MNPs were prepared by adapting an impregnation method, without using surface capping reagents and characterized (transmission electron microscopy, x-ray diffraction and Fourier-transform infrared spectroscopy) with their in vitro cytotoxicity assessed, followed by imaging assessments. Results: The contrast-enhancing ability of Au*MNPs was shown to be concentration-dependent across MR, CT and IVUS imaging. The Au content of the Au*MNP led to evident increases of the IVUS signal. Conclusion: We demonstrated that Au*MNPs showed concentration-dependent contrast-enhancing ability in MRI and CT imaging, and for the first-time in IVUS imaging due to the Au content. These Au*MNPs are promising toward solidifying tri-modal imaging-based theragnostics.

Pathogenic Mannheimia haemolytica Invades Differentiated Bovine Airway Epithelial Cells.

The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) and is responsible for significant economic losses to livestock industries worldwide. Healthy cattle are frequently colonized by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a 5-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognized mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonization and infection of the bovine respiratory tract.

Carboxymethyl cellulose coated magnetic nanoparticles transport across a human lung microvascular endothelial cell model of the blood-brain barrier.

Sustained and safe delivery of therapeutic agents across the blood-brain barrier (BBB) is one of the major challenges for the treatment of neurological disorders as this barrier limits the ability of most drug molecules to reach the brain. Targeted delivery of the drugs used to treat these disorders could potentially offer a considerable reduction of the common side effects of their treatment. The preparation and characterization of carboxymethyl cellulose (CMC) coated magnetic nanoparticles (Fe3O4@CMC) is reported as an alternative that meets the need for novel therapies capable of crossing the BBB. In vitro assays were used to evaluate the ability of these polysaccharide coated biocompatible, water-soluble, magnetic nanoparticles to deliver drug therapy across a model of the BBB. As a drug model, dopamine hydrochloride loading and release profiles in physiological solution were determined using UV-Vis spectroscopy. Cell viability tests in Human Lung Microvascular Endothelial (HLMVE) cell cultures showed no significant cell death, morphological changes or alterations in mitochondrial function after 24 and 48 h of exposure to the nanoparticles. Evidence of nanoparticle interactions and nanoparticle uptake by the cell membrane was obtained by electron microscopy (SEM and TEM) analyses. Permeability through a BBB model (the transwell assay) was evaluated to assess the ability of Fe3O4@CMC nanoparticles to be transported across a densely packed HLMVE cell barrier. The results suggest that these nanoparticles can be useful drug transport and release systems for the design of novel pharmaceutical agents for brain therapy.

Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface.

There is an urgent need to develop improved, physiologically-relevant in vitro models of airway epithelia with which to better understand the pathological processes associated with infection, allergies and toxicological insults of the respiratory tract of both humans and domesticated animals. In the present study, we have characterised the proliferation and differentiation of primary bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface (ALI) at three-day intervals over a period of 42 days from the introduction of the ALI. The differentiated BBEC model was highly representative of the ex vivo epithelium from which the epithelial cells were derived; a columnar, pseudostratified epithelium that was highly reflective of native airway epithelium was formed which comprised ciliated, goblet and basal cells. The hallmark defences of the respiratory tract, namely barrier function and mucociliary clearance, were present, thus demonstrating that the model is an excellent mimic of bovine respiratory epithelium. The epithelium was fully differentiated by day 21 post-ALI and, crucially, remained healthy and stable for a further 21 days. Thus, the differentiated BBEC model has a three-week window which will allow wide-ranging and long-term experiments to be performed in the fields of infection, toxicology or general airway physiology.

Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface.

Respiratory tract infections are of significant concern in the agriculture industry. There is a requirement for the development of well-characterised in vitro epithelial cell culture models in order to dissect the diverse molecular interactions occurring at the host-pathogen interface in airway epithelia. We have analysed key factors that influence growth and differentiation of ovine tracheal epithelial cells in an air-liquid interface (ALI) culture system. Cellular differentiation was assessed at 21 days post-ALI, a time-point which we have previously shown to be sufficient for differentiation in standard growth conditions. We identified a dose-dependent response to epidermal growth factor (EGF) in terms of both epithelial thickening and ciliation levels. Maximal ciliation levels were observed with 25 ng ml-1 EGF. We identified a strict requirement for retinoic acid (RA) in epithelial differentiation as RA exclusion resulted in the formation of a stratified squamous epithelium, devoid of cilia. The pore-density of the growth substrate also had an influence on differentiation as high pore-density inserts yielded higher levels of ciliation and more uniform cell layers than low pore-density inserts. Differentiation was also improved by culturing the cells in an atmosphere of sub-ambient oxygen concentration. We compared two submerged growth media and observed differences in the rate of proliferation/expansion, barrier formation and also in terminal differentiation. Taken together, these results indicate important differences between the response of ovine tracheal epithelial cells and other previously described airway epithelial models, to a variety of environmental conditions. These data also indicate that the phenotype of ovine tracheal epithelial cells can be tailored in vitro by precise modulation of growth conditions, thereby yielding a customisable, potential infection model.

Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells.

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-tagged nanoparticles offer as novel therapeutics in regenerative medicine.

Development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract.

Cattle are subject to economically-important respiratory tract infections by various bacterial and viral pathogens and there is an urgent need for the development of more realistic in vitro models of the bovine respiratory tract to improve our knowledge of disease pathogenesis. In the present study, we have optimized the culture conditions in serum-free medium that allow bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface to differentiate into a three-dimensional epithelium that is highly representative of the bovine airway. Epidermal growth factor was required to trigger both proliferation and differentiation of BBECs whilst retinoic acid was also essential for mucociliary differentiation. Triiodothyronine was demonstrated not to be important for the differentiation of BBECs. Oxygen concentration had a minimal effect although optimal ciliation was achieved when BBECs were cultured at 14% oxygen tension. Insert pore-density had a significant effect on the growth and differentiation of BBECs; a high-pore-density was required to trigger optimum differentiation. The established BBEC model will have wide-ranging applications for the study of bacterial and viral infections of the bovine respiratory tract; it will contribute to the development of improved vaccines and therapeutics and will reduce the use of cattle in in vivo experimentation.

Mesenchymal stem cell-derived extracellular vesicles may promote breast cancer cell dormancy.

Disseminated breast cancer cells have the capacity to metastasise to the bone marrow and reside in a dormant state within the mesenchymal stem cell niche. Research has focussed on paracrine signalling factors, such as soluble proteins, within the microenvironment. However, it is now clear extracellular vesicles secreted by resident mesenchymal stem cells into this microenvironment also play a key role in the initiation of dormancy. Dormancy encourages reduced cell proliferation and migration, while upregulating cell adhesion, thus retaining the cancer cells within the bone marrow microenvironment. Here, MCF7 breast cancer cells were treated with mesenchymal stem cell-derived extracellular vesicles, resulting in reduced migration in two-dimensional and three-dimensional culture, with reduced cell proliferation and enhanced adhesion, collectively supporting cancer cell dormancy.

Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface.

The respiratory tract and lungs are subject to diverse pathologies with wide-ranging implications for both human and animal welfare. The development and detailed characterization of cell culture models for studying such forms of disease is of critical importance. In recent years the use of air-liquid interface (ALI)-cultured airway epithelial cells has increased markedly, as this method of culture results in the formation of a highly representative, organotypic in vitro model system. In this study we have expanded on previous knowledge of differentiated ovine tracheal epithelial cells by analysing the progression of differentiation over an extensive time course at an ALI. We observed a pseudo-stratified epithelium with ciliation and a concurrent increase in cell layer thickness from 9 days post-ALI with ciliation approaching a maximum level at day 24. A similar pattern was observed with respect to mucus production with intensely stained PAS-positive cells appearing at day 12. Ultrastructural analysis by SEM confirmed the presence of both ciliated cells and mucus globules on the epithelial surface within this time-frame. Trans-epithelial electrical resistance (TEER) peaked at 1049 Ω × cm2 as the cell layer became confluent, followed by a subsequent reduction as differentiation proceeded and stabilization at ~200 Ω × cm2. Importantly, little deterioration or de-differentiation was observed over the 45 day time-course indicating that the model is suitable for long-term experiments.

Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence.

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies.

The influence of particle size and static magnetic fields on the uptake of magnetic nanoparticles into three dimensional cell-seeded collagen gel cultures.

Over recent decades there has been and continues to be major advances in the imaging, diagnosis and potential treatment of medical conditions, by the use of magnetic nanoparticles. However, to date the majority of cell delivery studies employ a traditional 2D monolayer culture. This article aims to determine the ability of various sized magnetic nanoparticles to penetrate and travel through a cell seeded collagen gel model, in the presence or absence of a magnetic field. Three different sized (100, 200, and 500 nm) nanoparticles were employed in the study. The results showed cell viability was unaffected by the presence of nanoparticles over a 24-h test period. The initial uptake of the 100 nm nanoparticle into the collagen gel structure was superior compared to the larger sized nanoparticles under the influence of a magnetic field and incubated for 24 h. Interestingly, it was the 200 nm nanoparticles, which proved to penetrate the gel furthest, under the influence of a magnetic field, during the initial culture stage after 1-h incubation.

Formation of octapod MnO nanoparticles with enhanced magnetic properties through kinetically-controlled thermal decomposition of polynuclear manganese complexes.

Polynuclear manganese complexes are used as precursors for the synthesis of manganese oxide nanoparticles (MnO NPs). Altering the thermal decomposition conditions can shift the nanoparticle product from spherical, thermodynamically-driven NPs to unusual, kinetically-controlled octapod structures. The resulting increased surface area profoundly alters the NP's surface-dependent magnetism and may have applications in nanomedicine.

Elucidating the function of penetratin and a static magnetic field in cellular uptake of magnetic nanoparticles.

Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake. Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake.

Design of multifunctional gold nanoparticles for in vitro and in vivo gene silencing.

Over the past decade, the capability of double-stranded RNAs to interfere with gene expression has driven new therapeutic approaches. Since small interfering RNA (siRNAs, 21 base pair double-stranded RNA) was shown to be able to elicit RNA interference (RNAi), efforts were directed toward the development of efficient delivery systems to preserve siRNA bioactivity throughout the delivery route, from the administration site to the target cell. Here we provide evidence of RNAi triggering, specifically silencing c-myc protooncogene, via the synthesis of a library of novel multifunctional gold nanoparticles (AuNPs). The efficiency of the AuNPs is demonstrated using a hierarchical approach including three biological systems of increasing complexity: in vitro cultured human cells, in vivo invertebrate (freshwater polyp, Hydra ), and in vivo vertebrate (mouse) models. Our synthetic methodology involved fine-tuning of multiple structural and functional moieties. Selection of the most active functionalities was assisted step-by-step through functional testing that adopted this hierarchical strategy. Merging these chemical and biological approaches led to a safe, nonpathogenic, self-tracking, and universally valid nanocarrier that could be exploited for therapeutic RNAi.

Working together: the combined application of a magnetic field and penetratin for the delivery of magnetic nanoparticles to cells in 3D.

Nanoparticles (NPs) are currently being developed as vehicles for in vivo drug delivery. Two of the biggest barriers facing this therapy are the site-specific targeting and consequent cellular uptake of drug-loaded NPs(1). In vitro studies in 2D cell cultures have shown that an external magnetic field (MF) and functionalization with cell-penetrating peptides (CPPs) have the capacity to overcome these barriers. This study aimed to investigate if the potential of these techniques, which has been reported in 2D, can be successfully applied to cells growing in a 3D environment. As such, this study provides a more realistic assessment of how these techniques might perform in future clinical settings. The effect of a MF and/or penetratin attachment on the uptake of 100 and 200 nm fluorescent iron oxide magnetic NPs (mNPs) into a fibroblast-seeded 3D collagen gel was quantified by inductively coupled plasma mass spectrometry. The most suitable mNP species was further investigated by fluorescence microscopy, histology, confocal microscopy, and TEM. Results show that gel mNP uptake occurred on average twice as fast in the presence of a MF and up to three times faster with penetratin attachment. In addition, a MF increased the distance of mNP travel through the gel, while penetratin increased mNP cell localization. This work is one of the first to demonstrate that MFs and CPPs can be effectively translated for use in 3D systems and, if applied together, will make excellent partners to achieve therapeutic drug delivery in vivo.

Influence of both a static magnetic field and penetratin on magnetic nanoparticle delivery into fibroblasts.

AIM: With regards to nanoparticles, all biomedical applications require cellular uptake, which to date remains a hurdle to further progress. This study aims to compare both the attractive force of a static magnetic field and the cell penetrating capability of penetratin; two techniques currently employed to enhance cell uptake. MATERIALS & METHODS: Fluorescent magnetic nanoparticles were functionalized with penetratin and cells were challenged with or without the particles in the presence/absence of a static magnetic field (350 mT). Following analysis of the magnetic field applied, cellular uptake and behavior was assessed in terms of fluorescence microscopy, clathrin and caveolin levels, scanning electron microscopy and transmission electron microscopy. RESULTS: Modeling of the field applied demonstrated varying field patterns across the cell culture area, reflected by higher particle uptake at higher field strengths. Both penetratin and the magnetic field increased cell uptake with penetratin proving more efficient. Interestingly, the magnetic field stimulated clathrin-mediated endocytosis and subsequent particle uptake.

Grooved surface topography alters matrix-metalloproteinase production by human fibroblasts.

Extracellular matrix (ECM) remodelling is an essential physiological process in which matrix-metalloproteinases (MMPs) have a key role. Manipulating the manner in which cells produce MMPs and ECMs may enable the creation of a desired tissue type, i.e. effect repair, or the prevention of tissue invasion (e.g. metastasis). The aim of this project was to determine if culturing fibroblasts on grooved topography altered collagen deposition or MMP production. Human fibroblasts were seeded on planar or grooved polycaprolactone substrates (grooves were 12.5 µm wide with varying depths of 240 nm, 540 nm or 2300 nm). Cell behaviour and collagen production were studied using fluorescence microscopy and the spent culture medium was assessed using gel zymography to detect MMPs. Total collagen deposition was high on the 240 nm deep grooves, but decreased as the groove depth increased, i.e. as cell contact guidance decreased. There was an increase in gelatinase on the 2300 nm deep grooved topography and there was a difference in the temporal expression of MMP-3 observed on the planar surface compared to the 540 nm and 2300 nm topographies. These results show that topography can alter collagen and MMP production. A fuller understanding of these processes may permit the design of surfaces tailored to tissue regeneration e.g. tendon repair.

The effect of static magnetic fields and tat peptides on cellular and nuclear uptake of magnetic nanoparticles.

Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field.

Cell-generated forces influence the viability, metabolism and mechanical properties of fibroblast-seeded collagen gel constructs.

The aim of this study was to investigate the influence of the endogenous forces generated by fibroblast-mediated contraction, using four individual collagen gel models that differed with respect to the ability of the cells to contract the gel. Human neonatal dermal fibroblasts were seeded in type I collagen and the gels were cast in a racetrack-shaped mould containing a removable central island. Two of the models were mechanically stressed (20 mm and 10 mm), as complete contraction was prevented by the presence of a central island. The central island was removed in the third model (released) and the final model was cast in a Petri dish and detached, allowing full multi-axial contraction (SR). Cell viability was maintained in the 10 mm, released and SR models over a 6 day culture period but localized regions of cell death were evident in the 20 mm model. Cell and collagen alignment was developed in the 20 mm and 10 mm models and to a lesser extent in the released model, but was absent in the SR model. Cell proliferation and collagen synthesis was lower in the 20 mm model compared to the other systems and there was evidence of enhanced matrix metalloproteinase production. The mechanical properties of the 20 mm model system were inferior to the 10 mm and released systems. The 10 mm model system induced a high level of cell and matrix orientation and may, therefore, represent the best option for tissue-engineered ligament repair involving an orientated fibroblast-seeded collagen gel.