Bio-electrospraying embryonic stem cells: interrogating cellular viability and pluripotency.

Bio-electrospraying, a recently discovered, direct electric field driven cell engineering process, has been demonstrated to have no harmful effects on treated cells at a molecular level. Although several cell types from both immortalized and primary cultures have been assessed post-treatment as a function of time in comparison to controls, the protocol has yet to be applied on embryonic stem cells. This is most important if bio-electrosprays are to further their applicability, in particular with regard to tissue engineering and regenerative medicine, where embryonic stem cells play a fundamental role. In the study presented herein the chosen stem cells are mouse embryonic stem (ES) cells. Hence, these first examples where embryonic stem cells have been jetted by way of bio-electrosprays, demonstrate the cellular viability and the cell's pluripotency indistinguishable when comparing those post-treated cells with their respective controls.

Part-time working: full-time professionalism.

This one-day conference brought together around 100 doctors from a wide range of specialties and at different stages of their careers to examine areas that are of increasing importance to the profession as a whole. Information gathered and imparted to institutions such as the postgraduate deaneries, royal colleges and specialist societies will add to the impetus for cultural and organisational changes so that the real potential of doctors working less than full time is not lost to the economy, NHS or patients.

A hybrid bio-jetting approach for directly engineering living cells.

This paper reports developments on a hybrid cell-engineering protocol coupling both bio-electrosprays and aerodynamically assisted bio-jets for process-handling living cells. The current work demonstrates the ability to couple these two cell-jetting protocols for handling a wide range of cells for deposition. The post-treated cells are assessed for their viability by way of flow cytometry, which illustrates a significant population of viable cells post-treatment in comparison to those controls. This work is the first example of coupling these two protocols for the process handling of living cells. The hybrid protocol demonstrates the achievement of stable cone jetting of a cellular suspension in the single-needle configuration which was previously unachieved with single-needle bio-electrosprays. Furthermore the living cells explored in these investigations expressed GFP, thus demonstrating the ability to couple gene therapy with this hybrid protocol. Hence, this approach could one day be explored for building biologically viable tissues incorporating a therapeutic payload for combating a range of cellular/tissue-based pathologies.

Receptor-targeted nanocomplexes optimized for gene transfer to primary vascular cells and explant cultures of rabbit aorta.

We have developed new, synthetic vector formulations that display high efficiency of gene transfer to vascular cells and tissues. The formulations comprise cationic liposomes and cationic, receptor-targeting peptides that self assemble on mixing with plasmid DNA into receptor-targeted nanocomplexes (RTNs). One such RTN formulation was optimal for transfection of primary smooth muscle cells (LYD-1), while a second was optimal for transfection of rabbit aortic explants (LYD-2). In both RTNs, the peptide was a 16-lysine motif linked to the targeting sequence CYGLPHKFCG via a short spacer sequence. The major difference between LYD-1 and LYD-2 lay in the cationic lipid component, where LYD-1 contained ditetradecyl trimethyl ammonium (DTDTMA), an unsaturated, cationic lipid with a 14-carbon alkyl tail, whereas LYD-2 contained 2,3-dioleyloxypropyl-1-trimethyl ammonium chloride (DOTMA), a cationic lipid with an 18-carbon unsaturated alkyl tail. LYD-2 transfections of aortic explants were effective with incubations performed at room temperature for as little as 30 minutes, with either saline or glucose-based solutions. Transgene expression in the explants peaked at 5 days and persisted for 14 days. The kinetics of transfected gene expression, along with the efficacy of transfection with short incubation times, indicate that these new formulations may be useful tools in the development of molecular therapies for cardiovascular diseases.

Bio-protocols for directly forming active encapsulations containing living primary cells.

Electrosprays and electrospinning were recently pioneered for directly handling living cells. These recent discoveries are now widely referred to as "bio-electrosprays" and "cell electrospinning", which have been demonstrated as having tremendous applicability to the life sciences in regenerative and therapeutic medicine. In the current work, we report our developmental studies with these protocols as submerged entities with primary rabbit aorta smooth muscle cells for generating cell-bearing encapsulations, which demonstrate proof-of-concept for a plethora of biomedical applications. Cell viability of the post-treated cells was assessed in comparison with two controls by way of flow cytometry over a three week period, establishing a viable cellular population >70%. Hence these investigations demonstrate the ability to explore these electrified encapsulating approaches for directly forming biologically viable emulsions, which could potentially be exploited from mechanisms for cancer therapy, hormone, and diabetic treatment to applications with cosmetics. Therefore, these studies elucidate the strong implications these bio-protocols have to offer the life sciences.

A unique physical-chemistry approach for fabricating cell friendly surfaces.

Recent interests in the fabrication of bio/cell-friendly surfaces are consistently gaining much scientific coverage as these methods could be explored as novel regenerative and therapeutic medicinal protocols. Essentially two main components govern this aspect, the processing methodology possessing the required robustness to fabricate a wide range of materials and, not least, the synthesised materials that need to be cell-compatible both in the short and long term after processing. In the study reported here we have combined one such robust jetting approach with a specially formulated siloxane sol. This has several unique properties in itself, and these have been demonstrated here to have a positive effect on the seeded cells. The current work demonstrates that this approach has great promise as a novel methodology for surface engineering for a wide range of applications spanning the physical to the life science areas of research.

Cardiac 82Rubidium PET/CT: initial European experience.

PURPOSE: Myocardial perfusion with PET/CT has advantages over conventional SPECT. We describe our initial European experience using (82)Rubidium-PET/CT, as part of a clinical myocardial perfusion service. METHODS: We studied the first 100 patients (64 male; 36 female, mean age = 60: SD +/-12.5y, mean body mass index = 30: SD +/-6.9kg/m( 2 )) who underwent (82)Rubidium cardiac PET/CT in our institution. Thirty patients had recently undergone coronary angiography. Patients underwent imaging during adenosine infusion and at rest. Images were acquired over 5 minutes using a GE-PET/CT instrument. Image quality was described as good, adequate or inadequate. Images were reported patient-by-patient by a minimum of five nuclear medicine physicians. A segment-by-segment analysis (17-segment model) was also performed. RESULTS: Image quality was good in 77%, adequate 23% and inadequate 0%. There was no statistical difference in image quality between obese and non-obese patients (Fisher's exact test, p = 0.2864). 59% had normal perfusion studies, 29% had inducible ischaemia, 12% had myocardial infarction (11% with super added ischaemia). There was reduced (82)Rubidium uptake in 132/1700 segments during stress. There was reduced (82)Rubidium uptake at rest in 42/1700 segments. The (82)Rubidium PET/CT findings were consistent with the angiographic findings in 28/30 cases. CONCLUSION: We show that, even from initial use of (82)Rubidium, it is possible to perform myocardial perfusion studies quickly with good image quality, even in the obese. The PET findings correlated well in the third of the cases where angiography was available. As such, (82)Rubidium cardiac PET/CT is likely to be an exciting addition to the European nuclear physician/ cardiologist's radionuclide imaging arsenal.

Cell electrospinning highly concentrated cellular suspensions containing primary living organisms into cell-bearing threads and scaffolds.

AIMS: We recently pioneered the cell electrospinning of living cells as viable biological threads and scaffolds. In that study, we demonstrated the process with an immortalized human brain astrocytoma (1321N1, European Collection of Cell Cultures) cell line at a cell concentration of 10(6) cells/ml. The next stage was to demonstrate the ability to cell electrospin primary living cells at cell concentrations of 10(7) cells/ml (the highest-ever cell concentration threaded by any threading methodology). Furthermore, the post-threaded cells needed their viability assessed over a long period of time by way of flow cytometry, which accurately assesses the viable cell populations. MATERIALS & METHODS: In this work, we employ primary porcine vascular and rabbit aorta smooth-muscle cells prepared as cellular suspensions at cell concentrations of 10(7) cells/ml. The cell electrospinning device employs a coaxial needle arrangement that enables the flow of either highly concentrated cellular suspension in the inner needle while the outer needle accommodates the flow of a viscoelasticity medical-grade polydimethylsiloxane medium. Cell viability was assessed over a long timeframe by way of flow cytometry in comparison with controls. RESULTS & DISCUSSION: The work reported here demonstrates the ability to cell electrospin primary living organisms as highly concentrated cellular suspensions. The viable population of cells post-cell electrospinning are significant and remain viable over both the short and long term, as assessed by flow cytometry. CONCLUSION: Our work elucidates the ability to cell electrospin primary cells as highly concentrated cellular suspensions. The post-cell electrospun organisms are viable over long periods of time, demonstrating a significant active cell population when compared with controls.

Bio-electrosprays: a novel electrified jetting methodology for the safe handling and deployment of primary living organisms.

Electrohydrodynamic jetting (EHDJ) which is also known as electrosprays (ES) has recently been elucidated as a unique electrified biotechnique for the safe handling and deployment of living organisms. This high intensity electric field driven jetting methodology is now referred to as "bioelectrosprays" (BES). Previously these charged jets have only been shown to jet-process immortalized cells which have undergone expected cellular behavior when compared with control cells. In this paper we demonstrate the ability to jet process primary living organisms in the stable conejetting mode. Finally the viability of the bio-electrosprayed living organisms has been assessed employing a flow cytometry approach which forms the discussion in this paper. Our findings further establish BES as a competing biotechnique, which could be employed for the deposition of primary living organisms according to a predetermined active cellular architecture. One day this could be used for the fabrication of viable tissues and organs for repair or replacement. These advanced studies carried out on BES have direct widespread applications ranging from developmental biology to regenerative and therapeutic medicine, which are a few amongst several other areas of study within the life sciences.

Aerodynamically assisted bio-jets: the development of a novel and direct non-electric field-driven methodology for engineering living organisms.

We recently demonstrated the ability to use electrified jets under stable conditions for the generation of cell-bearing droplets to the formation of composite threads which are biologically active. Our studies established that processed cells were viable over several generations post-jetting and -threading. These harmless and successful techniques for jet-based cell handling to deployment for precision deposition have great potential and widespread applications in bioengineering and biotechnology. Nonetheless, our investigations into 'bio-electrosprays' and 'cell electrospinning' have elucidated these jets having direct applicability in regenerative and therapeutic medicine to studies in developmental biology. For these very reasons, jet methodologies having the capability to safely handle living organisms for drop and placing are increasingly gaining the interests of life scientists. We now demonstrate yet another technique (a non-electric field-driven approach, previously never explored with jetting living cells), possessing the ability to directly handle the processing of primary living organisms by means of the flow of a cell suspension within a needle placed in a pressure chamber in the presence of an applied pressure difference. The technique we introduce here is referred to as 'aerodynamically assisted bio-jets/-jetting' which is driven completely by aerodynamic forces applied over an exit orifice by way of a differential pressure. Our investigations present an operational window in which stable jetting conditions are achieved for the formation of a near-monodispersed distribution of cell-bearing droplets and droplet residues. Finally, the aerodynamically bio-jetted living primary organisms are assessed (over both short and long time points) for cellular viability by means of FACScan, a flow cytometry technology which quantifies the percentage of living and dead cells. These advanced biophysical and bioengineering studies elucidate the emergence of a non-electric field-driven bio-jetting technology which now joins the cell jetting race.

Pressure-assisted cell spinning: a direct protocol for spinning biologically viable cell-bearing fibres and scaffolds.

We recently pioneered the ability to directly electrospin living cells from which scaffolds to membranes were derived. This protocol, now widely referred to as 'cell electrospinning', is currently undergoing in-depth investigations where the post-treated cell's global gene expression to its sub-cellular components is being investigated for understanding any effects post-treating. Our motivation is to develop this method for the biomedical sciences, in particular for applications in regenerative and therapeutic medicine. In the current work, we unveil a direct cell spinning protocol which is non-electric field driven and which will compete directly with cell electrospinning. We referred to this processing method as 'pressure-assisted spinning' in our previous studies, where we demonstrated this route as an emerging micro/nanotechnology. In the current context, we refer to this processing protocol as 'pressure-assisted cell spinning' (PACS). Our developmental studies on PACS reported here show, for the first time, that this technique could be explored as an alternative approach to cell electrospinning. Pressure-assisted cell spinning now enters the direct biological scaffold to membrane formation league.

Application to vascular adventitia of a nonviral vector for TIMP-1 gene therapy to prevent intimal hyperplasia.

Somatic gene transfer continues to have potential for the study and therapy of cardiovascular disease. We have developed a modular, self-assembling, nonviral system consisting of Lipofectin, integrin-targeting peptides, and plasmid DNA (LID) and we have applied this to a model of vascular injury, rat carotid angioplasty. Marker gene studies identified transfection of adventitial cells after vector delivery to that layer. Human tissue inhibitor of metalloproteinase-1 (hTIMP-1) was tested as a therapeutic gene product after direct application to the exposed adventitial layer. Vascular LID.hTIMP-1 transfection was confirmed by polymerase chain reaction and gene expression by immunohistochemistry at 7 days. Neointimal areas were 0.160 +/- 0.078 and 0.225 +/- 0.052 mm(2) for LID.hTIMP-1-transfected (n = 14) and LID.pCI-transfected (n = 12) vessels, respectively, at 14 days, and 0.116 +/- 0.068 mm(2) (n = 14) and 0.194 +/- 0.095 mm(2) (n = 14), respectively, at 28 days, representing a 29 and 40% reduction in neointimal hyperplasia at 14 and 28 days, respectively, after balloon dilatation. Neointima-to-media ratios were similarly reduced. In addition, expansile remodeling after balloon injury was inhibited at 14 days, the area within the external elastic lamina being 0.50 +/- 0.02 and 0.61 +/- 0.02 mm(2) in LID.hTIMP-1- and LID.pCI-transfected arteries, respectively (p < 0.0005). We have demonstrated an effective system of therapeutic gene transfer, particularly targeting the arterial adventitia, where transfer of genes involved in matrix remodeling and cell migration may be useful.

Acute depressed mood as a trigger of acute coronary syndromes.

BACKGROUND: Some cases of acute coronary syndrome (ACS) may be triggered by emotional states such as anger, but it is not known if acute depressed mood can act as a trigger. METHODS: 295 men and women with a verified ACS were studied. Depressed mood in the two hours before ACS symptom onset was compared with the same period 24 hours earlier (pair-matched analysis), and with usual levels of depressed mood, using case-crossover methods. RESULTS: 46 (18.2%) patients experienced depressed mood in the two hours before ACS onset. The odds of ACS following depressed mood were 2.50 (95% confidence intervals 1.05 to 6.56) in the pair-matched analysis, while the relative risk of ACS onset following depressed mood was 4.33 (95% confidence intervals 3.39 to 6.11) compared with usual levels of depressed mood. Depressed mood preceding ACS onset was more common in lower income patients (p = .032), and was associated with recent life stress, but was not related to psychiatric status. CONCLUSIONS: Acute depressed mood may elicit biological responses that contribute to ACS, including vascular endothelial dysfunction, inflammatory cytokine release and platelet activation. Acute depressed mood may trigger potentially life-threatening cardiac events.

Exaggerated platelet and hemodynamic reactivity to mental stress in men with coronary artery disease.

OBJECTIVE: This study compared the effects of acute mental stress on cardiovascular and subjective responses and platelet activation in male patients with established coronary artery disease (CAD) and age-matched controls. METHODS: We assessed 17 male CAD patients aged 44 to 59 years and 22 healthy male controls. Blood pressure, heart rate, and hemodynamics were assessed before, during, and up to 2 hours after administration of color/word and mirror tracing tasks. Blood was sampled at baseline, after tasks, and at 30 and 75 minutes after stress, and platelet activation was assessed by measuring platelet-leukocyte aggregates (PLAs) using flow cytometry. RESULTS: CAD patients showed significantly greater systolic blood pressure stress responses than controls (mean increases of 43.9 and 28.3 mm Hg, adjusted for income, body mass index, waist/hip ratio, and medication), together with larger increases in heart rate (14.1 and 4.7 bpm) and cardiac index. Total peripheral resistance increased during the poststress recovery period in CAD patients but not in controls. PLAs increased with stress in both groups, but remained elevated at 75 minutes in CAD patients, returning to baseline in controls. Heart rate and cardiac index responses were correlated with increases in subjective stress and with depression ratings, whereas PLA responses were associated with ratings of task difficulty. CONCLUSION: Acute mental stress stimulated heightened cardiovascular responses in CAD patients, coupled with more prolonged platelet activation. These factors may contribute to plaque rupture and thrombogenesis, and partly mediate stress-induced triggering of acute coronary syndromes.

Angina pectoris: interventional therapies and treatment of restenosis.

Angina pectoris is a clinical syndrome of symptoms caused by myocardial ischaemia due to oxygen demand exceeding supply. The most common cause is coronary artery stenosis due to progressive atherosclerotic disease. Angina has a prevalence of approximately 5% and increases with age. Despite improvements in treatment there remains a yearly mortality of 2-3%. A major advance in the treatment of symptomatic angina was the introduction of percutaneous transluminal coronary angioplasty (PTCA). This initial enthusiasm was dampened by significant numbers developing symptomatic restenosis from vascular elastic recoil and neointimal hyperplasia (NI). The widespread introduction of stent deployment following the initial angioplasty reduced the rates of elastic recoil but failed to prevent NI and may actually stimulate it. Currently, there is much interest in mechanisms that alter cell proliferation thereby decreasing NI. Techniques include brachytherapy, photodynamic therapy and drug-eluting stents. Provisional data for these new stents, which slowly release medication that inhibits cell turnover, are very good with few occurrences of restenosis. Results from larger randomised trials are awaited.

High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system.

BACKGROUND: Gene therapy strategies for the treatment of vascular disease such as the prevention of post-angioplasty restenosis require efficient, non-toxic transfection of vascular cells. In vitro studies in these cells contribute to vector development for in vivo use and for the evaluation of genes with therapeutic potential. The aim of this project was to evaluate a novel synthetic vector consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), which combine to form the LID vector complex. METHODS: Cultures of porcine smooth muscle cells and endothelial cells were established and then transfected with the LID vector, using the reporter genes luciferase and green fluorescent protein and the metalloprotease inhibitor TIMP-1. RESULTS: The LID vector system transfected primary porcine vascular smooth muscle cells and porcine aortic endothelial cells with efficiency levels of 40% and 35%, respectively. By increasing the relative DNA concentration four-fold, incubation periods as short as 30 min achieved the same levels of luciferase transgene expression as 4 h incubations at lower DNA concentrations. The transfection did not affect cell viability as measured by their proliferative potential. Serum levels of up to 20% in the transfection medium had no adverse affect on the efficiency of transfer and gene expression in either cell type. Transfections with the cDNA for TIMP-1 produced protein levels that peaked at 130 ng/ml per 24 h and persisted for 14 days at 10 ng/ml per 24 h. CONCLUSION: This novel vector system has potential for studies involving gene transfer to cardiovascular cells in vitro and in vivo.