Patterned Cell Cultures For High Throughput Studies Of Cell Electrophysiology And Drug Screening Applications

Over the last decade, the field of tissue and bio-engineering has seen an increase in the development of in vitro high-throughput hybrid systems that can be used to understand cell function and behavior at the cellular and tissue levels. These tools would have a wide array of applications including for implants, drug discovery, and toxicology, as well as for studying cell developmental behavior and as disease models. Currently, there are a limited number of efficient, functional drug screening assays in the pharmacology industry and studies of cell-surface interactions are complicated and invasive. Most cell physiology studies are performed using conventional patch-clamp techniques or random networks cultured on silicon devices such as Microelectrode Arrays (MEAs) and Field Effect transistors (FETs). The objective of this study was to develop high-throughput in vitro platforms that could be used to analyze cell function and their response to various stimuli. Our hypothesis was that by utilizing surface modification to provide external guidance cues for various cell types and by controlling the cell environment in terms of culture conditions, we could develop an in vitro hybrid platform for sensing and testing applications. Such a system would not only give information regarding the surface effects on the growth and behavior of cells for implant development applications, but also allow for the study of vital cell physiology parameters like conduction velocity in cardiomyocytes and synaptic plasticity in neuronal networks. This study outlines the development of these in vitro high throughput systems that have varied applications ranging from tissue engineering to drug development. We have developed a simple and relatively high-throughput method in order to test the physiological effects of varying iii chemical environments on rat embryonic cardiac myocytes in order to model the degradation effects of polymer scaffolds. Our results, using our simple test system, are in agreement with earlier observations that utilized a complex 3D biodegradable scaffold. Thus, surface functionalization with self-assembled monolayers combined with histological/physiological testing could be a relatively high throughput method for biocompatibility studies and for the optimization of the material/tissue interface in tissue engineering. Traditional multielectrode extracellular recording methods were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; for example, to enable the measurement of conduction velocity, refractory period after action potentials or to create a functional reentry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, the patterning of cardiac myocyte monolayers increased the information content of traditional multielectrode measurements. Patterning techniques with self-assembled monolayers on microelectrode arrays were also used to study the physiological properties of hippocampal networks with functional unidirectional connectivity, developed to study the mono-synaptic connections found in the dentate gyrus. Results indicate that changes in synaptic connectivity and strength were chemically induced in these patterned hippocampal networks. This method is currently being used for studying long term potentiation at the cellular level. For this purpose, two cell patterns were optimized for cell migration onto the pattern as demonstrated by time lapse studies, and for iv supporting the best pattern formation and cell survival on these networks. The networks formed mature interconnected spiking neurons. In conclusion, this study demonstrates the development and testing of in vitro highthroughput systems that have applications in drug development, understanding disease models and tissue engineering. It can be further developed for use with human cells to have a more predictive value than existing complex, expensive and time consuming methods

Comparative evaluation of sealing ability, penetration and adaptation of a self etching pit and fissure sealant- stereomicroscopic and scanning electron microscopic analyses

Background: The efficacy of pit and fissure sealants in preventing occlusal caries is a well-established fact. Considering the difficulty in achieving strict isolation for a longer duration while treating the pediatric patients, a simplified procedure of sealant application is desirable. While, a self-etching sealant, Prevent Seal offers a quick procedure, the physical properties of this material haven?t been studied yet. Thus, this study was aimed to comparatively evaluate sealing ability, penetration and adaptation of a self-etching pit and fissure sealant and a conventional resin sealant. Material and Methods: This was an in vitro intergroup comparative study, which consisted of 2 groups- Group I (Conventional acid etch sealant, Clinpro) and Group II (Self etching sealant, Prevent Seal). Out of 32 selected teeth 16 were used to study microleakage, with the help of dye penetration test using Övrebö and Raadal criteria. Remaining 16 were used to evaluate sealant penetration and adaptation viz bubbles in the bottom of fissure, debris in the fissure, tags in the bottom of the fissure and tags at cuspal slopes and fissure entrance was done using stereomicroscope. Post stereomicroscopic evaluation 4 samples each were randomly chosen from both the groups and checked for etching pattern using Scanning electronic microscope. Results: The comparison of tested properties between the groups was done using Chi square test. There was no statistically significant difference observed when microleakage and sealant penetration / adaptation properties were compared between two groups (p=0.63 and p= 0.131, 0.131, 0.302, 0.106 respectively). No conclusive results could be withdrawn while etching patterns were compared between the groups (p=0.717). Conclusions: The self-etching sealant Prevent seal was found to have similar microleakage, sealant penetration and adaptation properties as conventional acid etch sealant

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Growth And Electrophysiological Properties Of Rat Embryonic Cardiomyocytes On Hydroxyl- And Carboxyl-Modified Surfaces

Biodegradable scaffolds such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) or poly(glycolic acid) (PGA) are commonly used materials in tissue engineering. The chemical composition of these scaffolds changes during degradation which provides a differential environment for the seeded cells. In this study we have developed a simple and relatively high-throughput method in order to test the physiological effects of this varying chemical environment on rat embryonic cardiac myocytes. In order to model the different degradation stages of the scaffold, glass coverslips were functionalized with 11-mercaptoundecanoic acid (MUA) and 11-mercapto-1-undecanol (MUL) as carboxyl- and hydroxyl-groups presenting surfaces, and with trimethoxysilylpropyldiethylenetriamine (DETA) and (3-aminopropyl) triethoxysilane (APTES) as controls. Embryonic cardiac myocytes formed beating islands on all tested surfaces, but the number of attached cells and beating patches was significantly lower on MUL compared to any of the other functionalized surfaces. Moreover, whole-cell patch-clamp experiments showed that the average length of action potentials generated by the beating-cardiac myocytes were significantly longer on MUL compared to the other surfaces. Our results, using our simple test system, are in basic agreement with earlier observations that utilized a complex 3D biodegradable scaffold. Thus, surface functionalization with self-assembled monolayers combined with histological/physiological testing could be a relatively high throughput method for biocompatibility studies and for the optimization of the material/tissue interface in tissue engineering. © 2008 VSP

Patterning Of Diverse Mammalian Cell Types In Serum Free Medium With Photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer-by-layer self-assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short-term stability and low-protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell-selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108-15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers

Patterned Cardiomyocytes On Microelectrode Arrays As A Functional, High Information Content Drug Screening Platform

Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional re-entry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes. © 2011 Elsevier Ltd

Temporal Characterization Of Neuronal Migration Behavior On Chemically Patterned Neuronal Circuits In A Defined In Vitro Environment

Directed control of neuronal migration, facilitating the correct spatial positioning of neurons, is crucial to the development of a functional nervous system. An understanding of neuronal migration and positioning on patterned surfaces in vitro would also be beneficial for investigators seeking to design culture platforms capable of mimicking the complex functional architectures of neuronal tissues for drug development as well as basic biomedical research applications. This study used coplanar self-assembled monolayer patterns of cytophilic, N-1[3-(trimethoxysilyly)propyl] diethylenetriamine (DETA) and cytophobic, tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (13F) to assess the migratory behavior and physiological characteristics of cultured neurons. Analysis of time-lapse microscopy data revealed a dynamic procedure underlying the controlled migration of neurons, in response to extrinsic geometric and chemical cues, to promote the formation of distinct two-neuron circuits. Immunocytochemical characterization of the neurons highlights the organization of actin filaments (phalloidin) and microtubules (β-tubulin) at each migration stage. These data have applications in the development of precise artificial neuronal networks and provide a platform for investigating neuronal migration as well as neurite identification in differentiating cultured neurons. Importantly, the cytoskeletal arrangement of these cells identifies a specific mode of neuronal migration on these in vitro surfaces characterized by a single process determining the direction of cell migration and mimicking somal translocation behavior in vivo. Such information provides valuable additional insight into the mechanisms controlling neuronal development and maturation in vitro and validates the biochemical mechanisms underlying this behavior as representative of neuronal positioning phenomena in vivo

Ethanolic extracts of Aloe vera and propolis as cavity disinfectants: An in vitro study

Introduction: Though a large number of agents including chlorhexidine have been used for cavity disinfection during restorative procedures, till date none has proved to be "ideal." Thus, there is a need for an alternative cavity disinfectant, which is safe, effective, and economic. Herbal extracts of Aloe vera and propolis have shown the potential to be used as cavity disinfectants but not much is known about them. The aim of this study was to evaluate and compare the antibacterial activity of ethanolic extracts of Aloe vera and propolis against S. mutans and L. acidophilus, along with assessing the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Materials and Methods: The study included three groups: 2% chlorhexidine solution (group I), ethanolic extract of Aloe vera (group II), and ethanolic extract of propolis (group III). An MIC test was carried out using tissue culture microplates and serial dilution technique and later to MBC, an aliquot of each incubated well with concentrations higher than MIC was subcultured on brain heart infusion (BHI) medium. Antibacterial assay was carried out using agar disk diffusion technique and zones of inhibition were determined. One-way analysis of variance (ANOVA) was used for intergroup comparison followed by post hoc Tukey′s test for groupwise comparison. Results: Group II and Group III showed bacteriostatic effect but no bactericidal effect against both S. mutans and L. acidophilus. There was a significant difference in the antibacterial activity against both the tested microorganisms among all the groups, the highest being in group I followed by group II and group III. Conclusions: Ethanolic extracts of Aloe vera and propolis are only bacteriostatic and their antibacterial efficacy is inferior to chlorhexidine against both S. mutans and L. acidophilus

Adrenergic Deficiency Leads To Impaired Electrical Conduction And Increased Arrhythmic Potential In The Embryonic Mouse Heart

To determine if adrenergic hormones play a critical role in the functional development of the cardiac pacemaking and conduction system, we employed a mouse model where adrenergic hormone production was blocked due to targeted disruption of the dopamine β-hydroxylase (Dbh) gene. Immunofluorescent histochemical evaluation of the major gap junction protein, connexin 43, revealed that its expression was substantially decreased in adrenergic-deficient (Dbh-/-) relative to adrenergic-competent (Dbh+/+ and Dbh+/-) mouse hearts at embryonic day 10.5 (E10.5), whereas pacemaker and structural protein staining appeared similar. To evaluate cardiac electrical conduction in these hearts, we cultured them on microelectrode arrays (8×8, 200μm apart). Our results show a significant slowing of atrioventricular conduction in adrenergic-deficient hearts compared to controls (31.4±6.4 vs. 15.4±1.7ms, respectively, p\u3c0.05). To determine if the absence of adrenergic hormones affected heart rate and rhythm, mouse hearts from adrenergic-competent and deficient embryos were cultured ex vivo at E10.5, and heart rates were measured before and after challenge with the β-adrenergic receptor agonist, isoproterenol (0.5μM). On average, all hearts showed increased heart rate responses following isoproterenol challenge, but a significant (p\u3c0.05) 225% increase in the arrhythmic index (AI) was observed only in adrenergic-deficient hearts. These results show that adrenergic hormones may influence heart development by stimulating connexin 43 expression, facilitating atrioventricular conduction, and helping to maintain cardiac rhythm during a critical phase of embryonic development. © 2012 Elsevier Inc