Browsing by Author "Raj, V"

Now showing 1 - 6 of 6
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    A Cytocompatible Poly(N-isopropylacrylamide-co-glycidylmethacrylate) Coated Surface as New Substrate for Corneal Tissue Engineering
    (JOURNAL OF BIOACTIVE AND COMPATIBLE POLYMERS, 2010) Joseph, N; Prasad, T; Raj, V; Kumar, PRA; Sreenivasan, K; Kumary, TV
    The conventional method of retrieving cells for tissue engineering to create three-dimensional functional tissues uses enzymes that may hamper cell viability and re-adhesion. Culturing cells on thermoresponsive surfaces of poly(N-isopropylacrylamide) (PNIPAAm) is a relatively new nondestructive method of creating in vitro tissues. In this study, PNIPAAm and glycidylmethacrylate (GMA)-based thermoresponsive copolymer N-isopropylacrylamide-co-glycidylmethacrylate (NGMA) were synthesized as a potential cell culture harvesting system for generating 3D synthetic tissues. The copolymer was characterized by differential scanning calorimetry, gel permeation chromatography, Fourier transform infrared spectroscopy, water contact angle, atomic force microscopy, and nuclear magnetic resonance spectroscopy. The NGMA-coated dishes were evaluated for cytotoxicity and cytocompatibility using L-929 cells. Primary rabbit corneal cultures established on NGMA surface were detached as an intact cell sheet with epithelial specific characteristics as well as maintenance of cell-cell and cell-extracellular matrix contact. The results confirmed the suitability of NGMA substrate for cell culture and temperature-induced cell sheet harvest. This is the first report on this copolymer formulation as a substrate for tissue engineering application. Hydrophobic GMA apart from modulating the lower critical solution temperature features the prospects of further modification, namely the incorporation of biomolecules through the epoxy groups.
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    A Novel Thermoresponsive Graft Copolymer Containing Phosphorylated HEMA for Generating Detachable Cell Layers
    (JOURNAL OF APPLIED POLYMER SCIENCE, 2010) Abraham, TN; Raj, V; Prasad, T; Kumar, PRA; Sreenivasan, K; Kumary, TV
    A novel polymeric formulation based on N-isopropylacrylamide (NIPAAm), methylmethacrylate (MMA), and phosphorylated hydroxylethyl methacrylate (Phosp-HEMA) was synthesized and characterized. NIPAAm was copolymerized with a known quantity of MMA to form a poly(NIPAAm-MMA) copolymer and was subsequently grafted with Phosp-HEMA by gamma irradiation to a total dose of 0.5 kGy. The thermoresponsive graft copolymer was characterized by differential scanning calorimetry, Fourier transform infrared spectroscopy, contact angle measurements, and energy dispersive X-ray analysis. The cytotoxicity of the graft copolymer analyzed using L-929 fibroblast cells showed noncytotoxic response. The cell adhesion on the graft copolymer was studied using rabbit corneal cells (SIRC) and human osteoblasts (HOS). The adhered cells were found to spread leading to the formation of cell layers. The cell layers with intact cell-cell and cell-extra cellular matrix contact were detached by lowering temperature below the lower critical solution temperature (29 degrees C) of the graft copolymer. The viability and morphology of the cells in detached cell sheets were assessed by live dead staining and environmental scanning electron microscopy, respectively. This interesting feature of cell adhesion to form cell layers and cell sheet retrieval is implicit to be due to the properties of phosphate moieties on thermoresponsive copolymer. To the authors knowledge there is no previous report on phosphate moiety containing thermo responsive polymeric formulations which can modulate cell adhesion and cell sheet retrieval. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 115: 52-62, 2010
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    In vitro binding of C-reactive protein on to chemically modified polymethyl methacrylate surfaces
    (SENSORS AND ACTUATORS B-CHEMICAL, 2007) Sreenivasan, K; Raj, V; Han, PR
    Phosphorylethanolamine (PEA), C-reactive protein (CRP) specific ligand was coupled onto the surface of polymethyl methacrylate (PMMA) surface. Initially PMMA surface was modified by grafting glycidyl methacrylate (GMA). The anchoring of PEA on to PMMA was achieved through the ring opening reaction of epoxide group of GMA. The modified surface was characterized using FT-IR, scanning electron microscopy, energy dispersive X-ray analysis and contact angle measurement. The modified surface was found to bind CRP The adsorbed protein was visualized by staining Coomassie blue. The quantification of the extent of adsorption was carried out using spectrophotometric measurement. The methodology appears to be simple and expandable further for the development of a sensing element for CRP. (c) 2007 Elsevier B.V. All rights reserved.
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    In vitro cytocompatibility evaluation of a thermoresponsive NIPAAm-MMA copolymeric surface using L929 cells
    (JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE, 2010) Varghese, VM; Raj, V; Sreenivasan, K; Kumary, TV
    Scaffold free tissue constructs are preferred in tissue engineering as they overcome all the problems associated with scaffolds. Stimuli responsive polymers enable generation of scaffold free multilayered tissue constructs which would in turn reduce the use of biomaterials in vivo. In this study, we investigated cytocompatibility and thermoresponsiveness of a copolymer of N-Isopropylacrylamide and Methyl Methacrylate. Thermoresponsive surfaces were prepared by coating tissue culture polystyrene with the copolymer solution in isopropanol. Mammalian fibroblast cells (L929 cells) readily adhered on the copolymer. The viability and cellular activity was ensured through Neutral red staining, MTT assay, Tritiated thymidine uptake assay and Immunofluorescent staining for cytoskeletal organisation. Incubation under lower critical solution temperature of copolymer resulted in intact detachment of cells. To conclude, in-house synthesized cytocompatible smart culture substrate intended for tissue engineering was developed using a cost effective and simple technique. Moreover, presence of methyl methacrylate in the copolymer reduced the lower critical solution temperature facilitating extended in vitro manipulation time. As the copolymer is insoluble in water, the copolymer could be polymerised without additional crosslinkers.
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    Selective estimation of C-reactive protein in serum using polymeric formulations without antibody
    (SENSORS AND ACTUATORS B-CHEMICAL, 2010) Raj, V; Hari, PR; Antony, M; Sreenivasan, K
    A new method without employing antibody for the estimation of C-reactive protein (CRP) in serum is presented. The method centres on the variation of fluorescence intensity of pair copolymers, containing a fluorophore (Fluoreseinamine isomer 1) and O-Phosphorylethanolamine (PEA), a ligand of CRP. We found that the fluorescence of fluorophore attached copolymer was quenched in the presence of copolymer conjugated with PEA. The fluorescence emission was, however, increased when CRP was added into the solution of the copolymers. The intensity of fluorescence increased linearly up to a concentration of 250 ng/mL of CRP and then attained a constant value which remains the same with additional quantity of CRP. This observation was assigned to the saturation of the binding sites. Other proteins namely, albumin, fibrinogen and globulin did not show any interference in the measurement. The method was used for the estimation of CRP in blood serum of patients reported to the cardiology and the data compared well with the quantity of CRP in the same samples estimated using immunoassay method. Our results suggest that the polymer pick up CRP selectively from a complex milieu like serum. The method is simple, cost effective and sensitive with a detection limit of 20 ng/mL. (C) 2010 Elsevier B.V. All rights reserved.
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    A Simple Method to Efficiently Record/ Capture Caenorhabditis elegans Locomotory Behaviours
    (Protocols.io, 2019-01) Raj, V; Thekkuveettil, A
    Caenorhabditis elegans is an excellent model to study animal chemotaxis and thermotaxis behaviours. These nematodes have highly predictable behaviour pattern towards olfactory cues. A complex chemosensory information processing, based on both temporal and spacial cues, is involved in chemotaxis behaviour and can modify its behaviour towards attractants as well as repellents (Ward., 1973; Colbert et al., 1995; Troemel et al., 1997). In chemotaxis assay, worms show unsurpassed behaviour with a pattern of movement based on concentration gradient in the assay plate (Saeki et al., 2001; Iino et al., 2009). Such behavioural patterns are highly intriguing because they give better understanding on how various neuronal signalling elicit such pattern of behaviour and how factors such as past experience of the animal, mutations affecting neuronal function, modify them. (Brenner., 1974; de Bono et al., 1998). Hence, behavioural assays have critical role in elucidating the alterations in neuronal activities in C. elegans. The standard chemotaxis assay estimates the movement pattern of C. elegans by tracking the course it takes in an agar plate containing a chemical gradient. This measurement requires recording the tracks over time in the plate (Buckingham et al., 2005; Yemini et al., 2011). Automated single worm tracker allows long term behavioural recording (Husson et al., 2005; Wang et al., 2013). The pattern of behaviour of animals in the assay plates shows there are significant alterations in patterns of movement like body bends and omega turns under experimental conditions. For a long term observation for such behaviour one needs to record the animals with least disturbances. Efficient recording often eliminates researcher’s bias and makes it easy to re-evaluate the results if needed (Piere-Shimoura et al., 1999; Hardaker LA et al., 2001; Baek et al., 2002). These recorded videos can be later processed in ImageJ for measuring the patterns. Though these recording can be done using a simple dissection microscopic system with camera, there is a major limitation that the light source is very close to the worm making series of artefacts in its behaviour, Here we report a simple setup to manually record and count these behavioural changes in worms. In this study we measured basal slowing response and enhanced slowing responses, the two different locomotory changes in response to food. Neuronal circuitry underlying these locomotory changes involves dopamine and serotonin (Sawin et al 2000).