Browsing by Author "Krishnan, Lissy K."
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Item A Biochemical Study on the Effect of Proteolysis of Beta-Thromboglobulin Proteins Released from Activated Platelets on Fibroblast Proliferation(PATHOPHYSIOLOGY OF HAEMOSTASIS AND THROMBOSIS, 2007)beta-Thromboglobulin (beta-TG) proteins are a heterogeneous group released from platelet alpha-granules on activation and have an effect on fibroblast migration and proliferation. We have previously reported the action of a metal-dependent protease on platelet-released proteins, which generates low-molecular-weight proteins that could be inhibited by ethylenediaminetetraacetic acid (EDTA). To understand the physiological significance of the breakdown of proteins after release, their effect on fibroblast proliferation in vitro was studied. Platelet releasates were obtained without and with EDTA inhibition designated as R1 and R2, respectively, and proteins were affinity purified for testing. Cell proliferation was measured using [(3)H]-thymidine assay. Both R1 and R2 showed maximum activity at 100 mu g/ml and R2 elicited significant proliferation compared to R1. When affinity-purified proteins were tested at 100 ng/ml, high-molecular-weight proteins showed significantly higher proliferation than low-molecular-weight proteins. We have shown that beta-TG is cleaved after being released from activated platelets, thereby becoming less mitogenic for fibroblasts. Copyright (C) 2010 S. Karger AG, BaselItem Advantages of hyaluronic acid as a component of fibrin sheet for care of acute wound(BIOLOGICALS, 2011)Skin injury is followed by accumulation of a fibrin based provisional matrix which normally drives the process of wound repair. Exogenous fibrin with extra cellular matrix (ECM) components can also favor the wound healing process. In a preliminary study we found that lyophilized fibrin sheet (FS) arrest bleeding from rabbit skin wound but it remained dry during the repair period and did not accelerate the healing process better than untreated control. In the current study, hyaluronic acid (HA) was incorporated into FS and the resultant HA-FS promoted water retention and improved wound healing process. Gross-morphology, histopathology and histomorphometry were employed to compare qualitative and quantitative difference of wound healing in treated group against controls. In experimental sites (HA-FS), re-epithelialization was completed by 14 days with neo-vascularization and deposition of wavy bundles of collagen in the treated sites. Rate of healing process was different in treated and untreated wounds and most striking difference was the appearance of appendages, sebaceous gland and hair follicle at some locations in HA-FS treated sites. Therefore, HA with fibrin can create an effective wound care matrix which promotes water retention and wound healing potential. (C) 2011 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.Item Bio-mimetic composite matrix that promotes endothelial cell growth for modification of biomaterial surface(JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A, 2007)The incidence of thrombogenesis and occlusion of cardiovascular implants is likely to be reduced by endothelial cell (EC) growth promotion on biornaterials used for device fabrication. However, proper signaling between the matrix proteins deposited on the device surface and the cells grown on it is a prime requirement for growth and function. It was demonstrated earlier that a composition of matrix proteins that include fibrin, fibronectin, gelatin, and growth factors maintain a steady proliferation potential and prolong the survival of endothelial cells in vitro. In this study, assessment of the same matrix to prevent EC from dedifferentiation during in vitro culture and to promote endothelialization of biornaterials used for fabrication of cardiovascular implants is carried out. Up/ down regulation of m-RNA expression for a prothrombotic molecule-plasminogen activator inhibitor (PAI), and two antithrombotic molecules- nitric oxide synthetase (eNOS) and tissue plasminogen activator (t-PA) are chosen as the indicators of cell dedifferentiation during cell culture and passaging. Immunostaining for vinculin and actin demonstrated that composite coating on biomaterials improves focal adhesion and cytoskeletal organization that increases the quality of EC grown on it. EC proliferation, measured by H-3-thymidine uptake, on all bare materials was poor and high incidence of cell apoptosis was noticed within 72 h in culture, whereas once coated with composite all materials showed good proliferation and survival. The results suggest that the designed composition of biomimetic adhesive proteins and growth factors is suitable for EC growth, survival, and functional integrity, thus making it suitable for cardiovascular tissue engineering that requires in vitro EC culture. (c) 2006 Wiley Periodicals, Inc.Item Design of Fibrin Matrix Composition to Enhance Endothelial Cell Growth and Extracellular Matrix Deposition for In Vitro Tissue Engineering(ARTIFICIAL ORGANS, 2009)Tissue-engineered blood vessel substitutes should closely resemble native vessels in terms of structure, composition, mechanical properties, and function. Successful cardiovascular tissue engineering requires optimization of in vitro culture environment that would produce functional constructs. The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system to render elasticity to blood vessel wall, whereas collagen is responsible for providing mechanical strength. The objective of this study was to understand the significance of various ECM components on endothelial cell (EC) growth and tissue generation. We demonstrate that, even though fibrin is a good matrix for EC growth, fibronectin is the crucial component of the fibrin matrix that enhances EC adhesion, spreading, and proliferation. Vascular EC growth factor is known to influence in vitro growth of EC, but, so far, ECM deposition in in vitro culture has not been reported. In this study, it is shown that incorporation of a mixture of hypothalamus-derived angiogenic growth factors with fibrin matrix enhances synthesis and deposition of insoluble elastin and collagen in the matrix, within 10 days of in vitro culture. The results suggest that a carefully engineered fibrin composite matrix may support EC growth, survival, and remodeling of ECM in vitro and impart optimum properties to the construct for resisting the shear stress at the time of implantation.Item Development of a Fibrin Composite-Coated Poly(epsilon-Caprolactone) Scaffold for Potential Vascular Tissue Engineering Applications(JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS, 2008)Poor cell adhesion, cytotoxicity of degradation products and lack of biological signals for cell growth, survival, and tissue generation are the limitations in the use of a biodegradable polymer scaffold for vascular tissue engineering. We have fabricated a hybrid scaffold by integrating physicochemical characteristics of poly(epsilon-caprolactone) (PCL) and biomimetic property of a composite of fibrin, fibronectin, gelatin, growth factors, and proteoglycans to improve EC growth on the scaffold. Solvent cast porous films of poly(epsilon-caprolactone) was prepared using PEG as a porogen. Porosity varied between 5 and 200 mu m, and FTIR spectroscopy confirmed structural aspects of PCL. Films kept in PBS for 60 days showed tensile strength and elongation matching native blood vessel. Slow degradation of the scaffold was demonstrated by gravimetric analysis and molecular weight determination. Human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on bare films were minimal. FTIR spectroscopy and environmental scanning electron microscopy (ESEM) of PCL-fibrin hybrid scaffold confirmed the presence of fibrin composite on PCL film. HUVEC was subsequently cultured on hybrid scaffold, and continuous EC lining was observed in 15 and 30 days of culture using ESEM. Results suggest that the new hybrid scaffold can be a suitable candidate for cardiovascular tissue engineering. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 87B: 570-579, 2008Item Glycosaminoglycans restrained in a fibrin matrix improve ECM remodelling by endothelial cells grown for vascular tissue engineering(JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, 2009)The success of a biocompatible vascular graft depends upon its mechanical attributes and postimplantation healing responses. Mechanical strength is a paramount issue because grafts placed in the arterial circulation must be capable of withstanding long-term haemodynamic stress without graft failure. Extracellular matrix (ECM) proteins that are deposited by the cells to remodel the environment play a major role in determining the construct stability and strength. A suitable scaffold that stimulates ECM deposition and remodelling by cells grown in vitro may generate tissues with normal function. The objective of this study was to prove that fibrin matrix composition can be modified with growth factors (GFs) and glycosaminoglycans (GAGs) to promote ECM remodelling coupled with endothelial cell (EC) growth. Effect of GFs and GAGs on ECM production and remodelling was studied separately and in combination. Matrices recovered after EC cultures were analysed after immunochemical staining and it was observed that GFs and GAGs influence collagen IV and elastin deposition. Quantitative PCR analysis of mRNA after specific periods of culture demonstrated significant upregulation of elastin and collagen expression in EC by combination of GFs and GAGS when compared to their individual effects. The results of experiments conducted with various combinations of GFs and GAGs show that a biomimetic approach of immobilization of signalling molecules in fibrin can upregulate ECM remodelling with simultaneous degradation of the fibrin matrix and deposition of collagen IV and elastin. Hence, this combination may be suitable for cardiovascular tissue generation in vitro. Copyright (C) 2009 John Wiley & Sons, Ltd.Item Growth factors upregulate deposition and remodeling of ECM by endothelial cells cultured for tissue-engineering applications(BIOMOLECULAR ENGINEERING, 2007)Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial for their long-term function. The interaction between cells and extra-cellular components is indispensable in determining cellular behavior in tissues and on biomaterials. The fibrin that contains fibronectin shows promise in most aspects as a tissue engineering scaffold, whereas, deposition of elastin and collagen by endothelial cells grown in the lumen of the construct is desirable to improve post implant retention, mechanical stability and vasoresponsiveness. So far there is no report on production of extra-cellular matrix (ECM) proteins, elastin and collagen by endothelial cells (EC) in in vitro culture conditions. In this study, we have used a biomimetic approach of providing multiple growth factors (GF) in the fibronectin (FN)-containing fibrin matrix to induce production of elastin and collagen by the endothelial cells for application in vascular tissue engineering. Deposition of elastin and collagens with matrix remodeling is demonstrated through qualitative analysis of the matrices that were recovered after growing cells on the initial fibrin-FN-GF matrix. Expressions of mRNA for both proteins were assessed by real time polymerase chain reaction (RT-PCR) to estimate the effects of multiple growth factor compositions. Marked deposition of elastin and collagen was evidenced by staining the recovered matrix after different culture intervals. Obviously, the biomimetic environment created by adding angiogenic and platelet growth factors in the fibrin-fibronectin-gelatin matrix can induce deposition of collagens and elastin by EC. (C) 2007 Elsevier B.V. All rights reserved.Item Increased platelet cholesterol and decreased percentage volume of platelets as a secondary risk factor for coronary artery disease(PATHOPHYSIOLOGY OF HAEMOSTASIS AND THROMBOSIS, 2007)Platelet hyperactivity is likely to contribute to the progression of atherogenesis and organized thrombus formation on vascular surfaces. The purpose of this study was to examine the effect of hypercholesterolemia on the cholesterol content of platelets, on platelet responsiveness and other platelet indices using platelets from 5 groups of age-matched subjects (n = 30 each), which includes healthy controls. All groups except controls had a high plasma lipid profile. While subjects in group I had only hyperlipidemia, those in groups II and III had hyperlipidemia in conjunction with diabetes mellitus and hypertension, respectively. The fourth group consisted of patients with confirmed coronary artery disease (CAD). The parameters studied include packed cell volume of platelets (platelet crit), platelet distribution width (PDW), platelet cholesterol and platelet aggregation in response to adenosine diphosphate and collagen. All the patient groups showed increased platelet aggregation (p < 0.05) and low platelet crit compared with controls (p < 0.05). In addition, platelet cholesterol was increased in patients with coronary disease, hyperlipidemia and diabetes mellitus (p < 0.05) but not in patients with hypertension (p > 0.05); PDW was high only in CAD (p < 0.05). A higher PDW indicated a prothrombotic tendency in CAD patients. Our data suggest that hyperlipidemia increases the lipid content in platelets and enhances their reactivity. Hyperactive platelets with increased platelet cholesterol may contribute to accelerated atherogenesis associated with CAD. Copyright (C) 2008 S. Karger AG, Basel.Item Phenotype gradation of human saphenous vein endothelial cells from cardiovascular disease subjects(ENDOTHELIUM-JOURNAL OF ENDOTHELIAL CELL RESEARCH, 2006)The highly organized histological architecture of the vascular wall and the specialized cellular phenotypes are perturbed in conditions such as atherosclerosis, restenosis, and hypertension. Alterations of endothelial cell (EC) phenotype in cardiovascular diseases (CVDs) as an effect of alteration of extracellular matrix (ECM) composition have not been well understood. In vitro study of EC phenotype is limited because they tend to dedifferentiate in subcultures. The objective of this study was to use in vitro cell culture on a biomimetic matrix model to characterize phenotype of human saphenous vein endothelial cells (HSVECs) harvested from CVD patients. Parameters studied were mRNA expression and synthesis of von Willebrand factor (vWF), plasminogen activation inhibitor (PAI), tissue plasminogen activator (t-PA), and endothelial nitric oxide synthetase (eNOS). Proliferation and apoptosis of HSVEC cultures obtained from eight different patients were compared on two matrices until passage 12. In early passages, both the prothombotic molecules vWF and PAI were overexpressed, whereas the antithrombotic molecules t-PA and eNOS were underexpressed. With increase in passage number, low expression of prothrombotic molecules and high expression of antithrombotic molecules were seen in cells on the model matrix. But when cells were grown on conventional gelatin-coated polystyrene, expression of prothrombotic molecules amplified further and antithrombotic molecules lessened with the progression of passages. On the model matrix HSVECs showed good proliferation rate and survival through several passages. It is demonstrated that matrix composition can influence switching of EC phenotypes into pro/antithrombotic states. This matrix model may be suitable to study the effect of exogenous factors on EC dysfunction with respect to CVD.Item Regulation of endothelial cell phenotype by biomimetic matrix coated on biomaterials for cardiovascular tissue engineering(ACTA BIOMATERIALIA, 2008)One major weakness that all cardiovascular replacements have in common is the lack of endothelial cell (EC) growth and post-implant remodeling of the device. The emerging field of tissue engineering focuses on the in vitro generation of functional organ replacements using living endothelial cells and other vascular cells for which nondegradable or biodegradable scaffold base materials are used. In this paper, it is demonstrated that some of the cardiovascular device materials in clinical use lack the ability to promote endothelial cell growth in vitro. We previously established a biomimetic matrix composition which supports the growth of human umbilical vein endothelial cells (HUVECs) while maintaining normal physiology in vitro. Here the effectiveness of the same coating to preserve the normal antithrombotic phenotype of endothelial cells grown on biomaterials was evaluated. The up/down-regulation of two prothrombotic and two antithrombotic molecules by HUVECs grown on bare material surfaces were compared with that on composite-coated materials. The suitability of this approach for blood-contacting applications was investigated by in vitro blood compatibility studies as recommended in ISO10993 part 4, by putting an EC-seeded surface in contact with human whole blood. It is demonstrated that EC-seeded bare material surfaces are prothrombotic, whereas surfaces pre-coated with biomimetic molecules facilitated maintenance of the normal EC phenotype and reduced the risk of platelet adhesion and activation of blood coagulation. The results presented here suggest that matrix composed of biomimetic adhesive proteins and growth factors is suitable for cardiovascular tissue engineering to improve biological function, irrespective of the material chosen to meet the mechanical properties of the device. (C) 2007 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.Item Silver Nanoparticle Impregnated Poly (epsilon-Caprolactone) Scaffolds: Optimization of Antimicrobial and Noncytotoxic Concentrations(TISSUE ENGINEERING PART A, 2011)Use of silver nanoparticles (SNPs) for control of implant-associated infection is a promising strategy, if optimum antimicrobial yet nontoxic dose to mammalian cells is identified. This study was done to determine essential quantity of SNPs, which stimulate antimicrobial activity without cytotoxicity, when immobilized on poly (e-caprolactone) (PCL) scaffold proposed for vascular tissue engineering. During SNP synthesis and scaffold preparation, nanoparticle aggregation was protected using poly (ethylene glycol). Transmission electron microscopy was used to characterize SNP size and to detect its mobilization from scaffold to culture medium. Antimicrobial property of the SNP and its dose response was tested using both Gram-positive and Gram-negative bacteria by zone of inhibition assay. Endothelial cells (ECs), the main cell type required for vascular tissue engineering, were grown on scaffolds to identify the nontoxic dose. After seeding EC on scaffolds, cell attachment, spreading, and viability/survival were detected using specific markers by flow cytometric/fluorescence microscopic analysis. Real-time polymerase chain reaction detected effect of SNPs on mRNA expression of selected EC-specific functional proteins. Results suggest that even devoid of antibiotics in the medium, 0.1% (w/w) SNP on PCL scaffold is antimicrobial while nontoxic to EC at cellular and molecular level once cultured on the SNP-PCL scaffold.Item Suitability of measurement of swirling as a marker of platelet shape change in concentrates stored for transfusion(PLATELETS, 2006)Platelet discoid shape is known to correlate with in vivo viability after transfusion. Measurement of shape change requires invasive sampling and laborious assays, which is difficult to perform in a blood transfusion center as a routine test for quality control of stored platelets. The objective of this study was to establish suitability of swirling assessment in stored platelet suspension as a routine test for quality check, by comparing platelet shape change measurement carried out in parallel. The study was done in two types of bags obtained from different manufactures (Groups 1 and 2). Platelet concentrates (PC) were stored for 120 h and samples drawn at 24-h intervals, optical analysis at 540nm was carried out to quantify shape change in response to an agonist adenosine diphosphate (ADP). The same bags were subjected to swirling assessment, by two blood bank personnel independently and graded as positive (+,++,+++) or as negative, based on the silky appearance of the suspension. Swirling negative platelets were prepared by storing platelets at 4 degrees C for 24 h and were compared with swirling positive platelets. Other parameters studied in the samples drawn at 24-h intervals were platelet count, mean platelet volume, and blood gases. Swirling assessment results correlated well with shape change measurement at each study period with a P value significant at 0.02 and 0.04 for group 1 and 2 bags, respectively. In the negative swirling controls, extent of shape change was lower than the extent in test bags, showing reduced capacity to respond to ADP at 4 degrees C. The results of the study indicate that by simple swirling measurements, stored PC can be monitored for loss of discoid shape before they are transfused. Gas tension and pH were with in permissible limits. Therefore, inspection of swirling can be a reliable method of quality control as it correlates with platelet function. The platelets that retain the discoid shape in vitro at the time of transfusion are expected to be functional in vivo.Item The antithrombotic and antimicrobial properties of PEG-protected silver nanoparticle coated surfaces(BIOMATERIALS, 2012)Cardiovascular implant-associated complications such as infection and thrombosis may be reduced by modification of device surfaces using antimicrobial and antithrombotic agents. Silver nanoparticles (SNPs) are well accepted for its broad-spectrum antimicrobial effect. A recent report suggested its antiplatelet effect also. So the hypothesis of this study is that polyethylene glycol (PEG) protected SNPs can be incorporated with biomaterials to attain dual properties; and by adjusting an optimum concentration, its cytotoxicity to tissues and cells can be prevented. To prove this, detailed study of PEG-SNP was done at three levels: (i) direct inhibitory effect on platelet activation, aggregation and biochemical pathways when PEG-SNP is added into platelet suspension; (ii) inhibition of platelet adhesion to PEG-SNP incorporated biological matrix and polymer scaffold and (iii) non-cytotoxic behavior of immobilized PEG-SNP in fibrin matrix. Inhibitory effects demonstrated are on: platelet function by aggregometry, exposure of activation and apoptosis markers by flow cytometry, biochemical pathway by malondealdehyde (MDA) estimation and protein phosphorylation by Western blot. Reduced platelet adhesion onto PEG-SNP incorporated scaffold is shown using scanning electron microscopy (SEM). Non-toxic behavior of endothelial cells (EC) and smooth muscle cells (SMC) grown on PEG-SNP-fibrin disc is shown by fluorescence microscopy and cell phenotype stability by real-time polymerase chain reaction (PCR). (c) 2012 Elsevier Ltd. All rights reserved.Item Vascular tissue construction on poly(e-caprolactone) scaffolds by dynamic endothelial cell seeding: effect of pore size(JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, 2012)In vitro tissue engineering for fabrication of small diameter vascular grafts probably undergoes a sequence of events similar to the in vivo angiogenesis process. In both cases endothelial cells (ECs) play the crucial role in generating a non-thrombogenic vessel lumen and stabilization of ECs in the lumen of new vessels requires the deposition of collagen IV and elastin. Shear stress is an important in vivo signal for inducing synthesis of extracellular matrix (ECM) components, collagen IV and elastin, which form the basement membrane in the case of new blood vessels. Stimulation of ECs may therefore produce collagen and elastin in the lumen of a polymeric scaffold during the vascular tissue-engineering process if appropriate biochemical and mechanical signals are presented. However, the morphology and physicochemical characteristics of polymer scaffolds may also be crucial for EC monolayer formation and ECM deposition. In this study, tubular scaffolds made of biodegradable poly(e-caprolactone) (PCL) with biomimetic fibrin-based coating were evaluated to compare the effects of pore sizes on surface coverage of ECs and synthesis of ECM under dynamic culture conditions. Actin was stained for identification of cells, while specific antibodies were used for locating collagen IV and elastin deposition on the scaffolds. It was found that dynamic seeding of ECs in the lumen stabilized the cells and aligned them along the direction of flow, with better deposition of insoluble elastin and collagen IV when similar to 75% of pores were < 24 mu m in diameter. In addition, monolayer on the e-PCL scaffolds with lower pore sizes was found to produce nitric oxide (NO), indicating a non-thrombogenic EC layer in the lumen. Copyright (c) 2011 John Wiley & Sons, Ltd.Item Vascular tissue generation in response to signaling molecules integrated with a novel poly(epsilon-caprolactone)-fibrin hybrid scaffold(JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, 2007)A major constraint in the use of biodegradable polymer scaffolds for vascular tissue engineering is poor cell adhesion and lack of signals for new tissue generation. The presence of extracellular matrix (ECM) within the scaffold is desirable for growth of endothelial cells and in vitro formation of remodelled vascular conduit. In this study, we have produced a hybrid scaffold by coating porous poly-caprolactone (PCL) film with biomimetic ECM components consisting of fibrin, gelatin, fibronectin, angiogenic growth factors and proteoglycans. Human umbilical vein endothelial cells (HUVECs) adhered, spread, proliferated and survived for long periods in culture on the hybrid scaffold. As compared to bare PCL, enhanced cell adhesion, spreading and cytoskeletal organization were demonstrated on the hybrid scaffold, using confocal microscopy of EC-actin stained with Texas red-conjugated phalloidin. Population doubling of endothelial cells (ECs) on the hybrid scaffold and bare scaffold was estimated as 42 h and 136 h, respectively, as assessed by a H-3-thymidine uptake method. Analysis of proliferating cell nuclear antigen (PCNA) also indicated low proliferation on bare scaffold. Flow cytometric analysis of annexin V-stained cells showed poor survival of ECs on bare PCL as compared to the hybrid scaffold. Deposition of-insoluble collagen and elastin was identified on the hybrid scaffold by cells recovered after 15 days and 30 days of EC culture, using fluorochrome-tagged specific antibodies and confocal microscopy, and the fluorescence intensity corresponding to elastin and collagen after 30 days was similar to that of 15 days. The results indicate that ECM deposition by endothelial cells is a regulated process without excesive accumulation after 30 days. Copyright (c) 2007 John Wiley & Sons, Ltd.