Lissy K Krishnan

Publications of Lissy K Krishnan

• The antithrombotic and antimicrobial properties of PEG-protected silver nanoparticle coated surfaces.

  Authors: V M Ragaseema, S Unnikrishnan, V Kalliyana Krishnan, Lissy K Krishnan

  Biomaterials. 04/2012; 33(11):3083-92.

  Cardiovascular implant-associated complications such as infection and thrombosis may be reduced by modification of device surfaces using antimicrobial and antithrombotic agents. Silver nanoparticlesCardiovascular 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).

• Vascular tissue construction on poly(ε-caprolactone) scaffolds by dynamic endothelial cell seeding: effect of pore size.

  Authors: Asha Mathews, Soumya Colombus, V Kalliyana Krishnan, Lissy K Krishnan

  Journal of tissue engineering and regenerative medicine. 07/2011;

  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 cellsIn 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(ε-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 ∼75% of pores were < 24 µm in diameter. In addition, monolayer on the ε-PCL scaffolds with lower pore sizes was found to produce nitric oxide (NO), indicating a non-thrombogenic EC layer in the lumen. Copyright © 2011 John Wiley & Sons, Ltd.

• Advantages of hyaluronic acid as a component of fibrin sheet for care of acute wound.

  Authors: T V Anilkumar, Jaseer Muhamed, Anumol Jose, Arun Jyothi, P V Mohanan, Lissy K Krishnan

  Biologicals : journal of the International Association of Biological Standardization. 02/2011; 39(2):81-8.

  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 alsoSkin 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.

• Silver nanoparticle impregnated poly (ɛ-caprolactone) scaffolds: optimization of antimicrobial and noncytotoxic concentrations.

  Authors: Ragaseema V Madhavan, Mathirappillil J Rosemary, Maya A Nandkumar, Kalliyana V Krishnan, Lissy K Krishnan

  Tissue engineering. Part A. 02/2011; 17(3-4):439-49.

  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 wasUse 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 (ɛ-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.

• Functional stability of endothelial cells on a novel hybrid scaffold for vascular tissue engineering.

  Authors: Divya Pankajakshan, Kalliyana Krishnan V, Lissy K Krishnan

  Biofabrication. 09/2010; 2(4):041001.

  Porous and pliable conduits made of biodegradable polymeric scaffolds offer great potential for the development of blood vessel substitutes but they generally lack signals for cell proliferation,Porous and pliable conduits made of biodegradable polymeric scaffolds offer great potential for the development of blood vessel substitutes but they generally lack signals for cell proliferation, survival and maintenance of a normal phenotype. In this study we have prepared and evaluated porous poly(ε-caprolactone) (PCL) integrated with fibrin composite (FC) to get a biomimetic hybrid scaffold (FC PCL) with the biological properties of fibrin, fibronectin (FN), gelatin, growth factors and glycosaminoglycans. Reduced platelet adhesion on a human umbilical vein endothelial cell-seeded hybrid scaffold as compared to bare PCL or FC PCL was observed, which suggests the non-thrombogenic nature of the tissue-engineered scaffold. Analysis of real-time polymerase chain reaction (RT-PCR) after 5 days of endothelial cell (EC) culture on a hybrid scaffold indicated that the prothrombotic von Willebrand factor and plasminogen activator inhibitor (PAI) were quiescent and stable. Meanwhile, dynamic expressions of tissue plasminogen activator (tPA) and endothelial nitric oxide synthase indicated the desired cell phenotype on the scaffold. On the hybrid scaffold, shear stress could induce enhanced nitric oxide release, which implicates vaso-responsiveness of EC grown on the tissue-engineered construct. Significant upregulation of mRNA for extracellular matrix (ECM) proteins, collagen IV and elastin, in EC was detected by RT-PCR after growing them on the hybrid scaffold and FC-coated tissue culture polystyrene (FC TCPS) but not on FN-coated TCPS. The results indicate that the FC PCL hybrid scaffold can accomplish a remodeled ECM and non-thrombogenic EC phenotype, and can be further investigated as a scaffold for cardiovascular tissue engineering.

• Effect of matrix composition on differentiation of nestin-positive neural progenitors from circulation into neurons.

  Authors: Anumol Jose, Lissy K Krishnan

  Journal of neural engineering. 06/2010; 7(3):036009.

  The human peripheral blood mononuclear cell has a mixture of progenitor cells with potential to differentiate into a wide range of lineages. The ability of hematopoietic tissue-derived adult stemThe human peripheral blood mononuclear cell has a mixture of progenitor cells with potential to differentiate into a wide range of lineages. The ability of hematopoietic tissue-derived adult stem cells to differentiate into neural progenitor cells offers an alternative to embryonic stem cells as a viable source for cell transplantation therapies to cure neurodegenerative diseases. This approach could lead to the use of autologous progenitors from blood circulation; however, due to the limited numbers available, in vitro cell expansion may be indispensable. In addition, for successful transplantation there is the requirement of a delivery matrix on which cells can survive and differentiate. In this context we carried out this study to identify a suitable biodegradable matrix on which progenitor cells can home, multiply and differentiate. We designed different compositions of the biomimetic matrix containing fibrin, fibronectin, gelatin, growth factors, laminin and hyaluronic acid. The attached cells expressed proliferation markers in initial periods of culture and between days 6 and 9 in culture they differentiated into neurons and/or astrocytes. The differentiation of progenitors into neurons and asterocyte on the composed matrix was established by morphological and immunochemical analysis. Flow cytometric analysis of cells in culture was employed to track development of neurons which expressed an early marker beta-tubulin3 and a terminal marker microtubule-associated protein-2 at a later culture period. In vitro experiments indicate that a highly specific niche consisting of various components of the extracellular matrix, including hyaluronic acid, promote cell homing, survival and differentiation.

• A biochemical study on the effect of proteolysis of beta-thromboglobulin proteins released from activated platelets on fibroblast proliferation.

  Authors: Resmi Ravindran, Lissy K Krishnan

  Pathophysiology of haemostasis and thrombosis. 03/2010; 36(6):285-9.

  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 previouslybeta-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 microg/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.

• Glycosaminoglycans restrained in a fibrin matrix improve ECM remodelling by endothelial cells grown for vascular tissue engineering.

  Authors: Pankajakshan Divya, Lissy K Krishnan

  Journal of tissue engineering and regenerative medicine. 06/2009;

  The success of a biocompatible vascular graft depends upon its mechanical attributes and post-implantation healing responses. Mechanical strength is a paramount issue because grafts placed in theThe success of a biocompatible vascular graft depends upon its mechanical attributes and post-implantation 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.

• Design of fibrin matrix composition to enhance endothelial cell growth and extracellular matrix deposition for in vitro tissue engineering.

  Authors: Divya Pankajakshan, Lissy K Krishnan

  Artificial organs. 02/2009; 33(1):16-25.

  Tissue-engineered blood vessel substitutes should closely resemble native vessels in terms of structure, composition, mechanical properties, and function. Successful cardiovascular tissue engineeringTissue-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.

• Development of a fibrin composite-coated poly(epsilon-caprolactone) scaffold for potential vascular tissue engineering applications.

  Authors: Divya Pankajakshan, Lizymol P Philipose, Minshiya Palakkal, Kalliyana Krishnan, Lissy K Krishnan

  Journal of biomedical materials research. Part B, Applied biomaterials. 07/2008;

  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 polymerPoor 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 mum, 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, 2008.

• Regulation of endothelial cell phenotype by biomimetic matrix coated on biomaterials for cardiovascular tissue engineering.

  Authors: Chennazhy Krishna Prasad, Lissy K Krishnan

  Acta biomaterialia. 02/2008; 4(1):182-91.

  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 engineeringOne 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.

• Growth factors upregulate deposition and remodeling of ECM by endothelial cells cultured for tissue-engineering applications.

  Authors: P Divya, P R Sreerekha, Lissy K Krishnan

  Biomolecular engineering. 01/2008; 24(6):593-602.

  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-cellularAppropriate 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 vaso-responsiveness. 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.

• Bio-mimetic composite matrix that promotes endothelial cell growth for modification of biomaterial surface.

  Authors: C Krishna Prasad, C V Muraleedharan, Lissy K Krishnan

  Journal of biomedical materials research. Part A. 04/2007; 80(3):644-54.

  The incidence of thrombogenesis and occlusion of cardiovascular implants is likely to be reduced by endothelial cell (EC) growth promotion on biomaterials used for device fabrication. However, properThe incidence of thrombogenesis and occlusion of cardiovascular implants is likely to be reduced by endothelial cell (EC) growth promotion on biomaterials 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 biomaterials 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 (3)H-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.

• Increased platelet cholesterol and decreased percentage volume of platelets as a secondary risk factor for coronary artery disease.

  Authors: Resmi Ravindran, Lissy K Krishnan

  Pathophysiology of haemostasis and thrombosis. 02/2007; 36(1):45-51.

  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 ofPlatelet 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.

• Suitability of measurement of swirling as a marker of platelet shape change in concentrates stored for transfusion.

  Authors: Jaisy Mathai, K R Resmi, P V Sulochana, S Sathyabhama, G Baby Saritha, Lissy K Krishnan

  Platelets. 10/2006; 17(6):393-6.

  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 aPlatelet 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 540 nm 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.

• Cultivation of endothelial progenitor cells on fibrin matrix and layering on dacron/polytetrafluoroethylene vascular grafts.

  Authors: Perumcherry Raman Sreerekha, Lissy K Krishnan

  Artificial organs. 05/2006; 30(4):242-9.

  Completely biological tissue-engineered vascular graft is an upcoming substitute for damaged blood vessel, but its clinical use is currently limited due to poor mechanical strength. Therefore, atCompletely biological tissue-engineered vascular graft is an upcoming substitute for damaged blood vessel, but its clinical use is currently limited due to poor mechanical strength. Therefore, at present, polymeric small-diameter vascular grafts lined with endothelial cells (ECs) to reduce graft thrombosis may be a more viable option. Successful construction of EC-seeded artificial grafts faces some challenges such as (i) retention of endothelial lining; and (ii) availability of differentiated autologous cells. Biomaterial surfaces that are modified by depositing extracellular matrix (ECM) components may stabilize cells in the lumen against forces of blood flow. Adult stem cells such as endothelial progenitor cells (EPCs) circulate in the blood and they usually attach to the exposed matrix at the injured blood vessel site. Depending on the signaling capabilities of ECM, cells may differentiate into ECs,, and if a similar composition of the matrix is provided in vitro, EPCs isolated from blood might get differentiated and thus autologous cells for tissue engineering may be obtained. In this in vitro study, ECM scaffold consisting of biomolecules such as fibrin, fibronectin, and gelatin along with growth factors is found to have supported differentiation of EPC into EC. Further, the ECM precoated on Dacron and polytetrafluoroethylene is found to have supported the formation of EC monolayer that synthesized nitric oxide, and resisted shear stress. Thus, biomimetic fibrin composite is found to be suitable not only to seed cells on currently available artificial grafts but also to obtain differentiated EC from EPC.

• Effect of passage number and matrix characteristics on differentiation of endothelial cells cultured for tissue engineering.

  Authors: Krishna Prasad Chennazhy, Lissy K Krishnan

  Biomaterials. 11/2005; 26(28):5658-67.

  Cells can sense the physical and chemical properties of artificial materials used as scaffolds for tissue engineering and regulate their behavior. Therefore, biomimetic and biospecific molecules areCells can sense the physical and chemical properties of artificial materials used as scaffolds for tissue engineering and regulate their behavior. Therefore, biomimetic and biospecific molecules are coated on materials to regulate function of cells on the tissue-engineered product. These bioactive molecules can be attached in a defined spectrum, concentration and spatial distribution in order to control adhesion, growth, viability, differentiation, and function of the cells. When autologous cells are used for tissue engineering, initially limited cells obtained may often need an amplification of cell number by passage in tissue culture before they are seeded on a biomaterial or scaffold. We have conducted this study to understand how the characteristics of bioactive molecule coating might affect proliferation, apoptosis and differentiation when endothelial cell (EC) is serially passaged. Proliferation was assessed by proliferating cell nuclear antigen (PCNA) staining along with counting of cells harvested from confluent monolayer. Apoptosis was assessed by Annexin V staining and differentiation by semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for von Willebrand factor (vWF) expression and quantification of its release using enzyme linked immunosorbant assay (ELISA), and thrombogenicity by comparing platelet adhesion to EC monolayer Dacron grafts (DG) with specific protein coating. The results indicate that ECs easily lose its proliferation potential when they are cultured repeatedly on gelatin, turn apoptotic and over express the prothrombotic protein- vWF. Whereas, when it is grown on a matrix composed of fibrin, fibronectin, gelatin and vascular EC growth factor (VEGF), the cells retained their ability to proliferate, remained viable and were relatively less thrombogenic, even when passage number progressed. It is concluded that if ECs are grown on the composite matrix that mimics natural vessel scaffold, the cell number can be amplified without affecting its normal physiological function and may be used to generate effective tissue-engineered cardiovascular constructs.

• Survival of endothelial cells in vitro on Paclitaxel-loaded coronary stents.

  Authors: C Krishna Prasad, K R Resmi, Lissy K Krishnan, Rajesh Vaishnav

  Journal of biomaterials applications. 04/2005; 19(4):271-86.

  Coronary stents that are developed for use with balloon angioplasty are known to cause acute occlusion and long-term stenosis. It is likely that a controlled release of drugs at the site of stentCoronary stents that are developed for use with balloon angioplasty are known to cause acute occlusion and long-term stenosis. It is likely that a controlled release of drugs at the site of stent implantation might inhibit the proliferation of vascular smooth muscle cells (VSMC) and reduce restenosis. However, if the drug is necrotic and affects cell survival near the implant, it may interrupt the local tissue regeneration. Different methods have been used for the immobilization of drugs with stents to get an effective concentration that inhibits cell proliferation. The objective of this study is to assess the effectiveness of Paclitaxel-loaded stents by immobilization with a biodegradable polymer, to inhibit cell proliferation. The cells used for the evaluation are human umbilical vein endothelial cells (HUVEC) and the proliferation rate of these cells on the drug-coated stent is compared against an uncoated stent for a 72-h period. Evaluations were also made to differentiate between cell apoptosis and necrosis to prove that the drug released is not deleterious to the surrounding tissue.While a similar initial cell adhesion is observed in bare and coated stents, the proliferation of HUVEC is negligible when grown on a drug-coated stent (p < 0.001). By specific staining techniques, the cells on the drug-coated stents are found to be apoptotic and not necrotic, throughout the evaluation period. In vitro leukocyte adhesion and platelet deposition on the drug-coated stents are found to be low when they are exposed to human blood and platelet-rich plasma (PRP), suggesting that the coated stents may not be thrombogenic in vivo. Therefore, drug coating of stents using the described technique may have a considerable promise for the prevention of neointimal proliferation, restenosis, and associated failure of angioplasty.

• Comparative evaluation of absorbable hemostats: advantages of fibrin-based sheets.

  Authors: Lissy K Krishnan, Mira Mohanty, P R Umashankar, Arthur Vijayan Lal

  Biomaterials. 12/2004; 25(24):5557-63.

  Bioactive hemostats and wound dressings consist of either inherently active materials or act as delivery vehicles which contain such materials. Fibrin is a natural hemostat and scaffold, guiding theBioactive hemostats and wound dressings consist of either inherently active materials or act as delivery vehicles which contain such materials. Fibrin is a natural hemostat and scaffold, guiding the direction of wound contraction and closure. In order to improve the ease of application of liquid fibrin glue, we have made a freeze-dried form of polymerized fibrin that supports hemostasis and wound healing. The bleeding from the middle ear artery of rabbits was found to be arrested instantaneously on application of fibrin sheets, even when the animal was heparinized systemically. As the fibrin sheet was found to be fragile, gelatin was incorporated to the sheet and thus the mechanical stability was improved without compromising the hemostatic effect. The efficacy of the fabricated fibrin and fibrin-gelatin sheets to seal traumatized rat liver was compared with commercially available hemostats, Abgel (cross-linked gelatin) and Surgicel (cross-linked cellulose). Tissue compatibility of all the hemostats was studied by analyzing the liver tissue 15 days after application. While the hemostatic effect was best with fibrin and fibrin-gelatin sheets, both Surgicel and Abgel were not capable of arresting the bleeding quickly. Gross analysis of tissue on the 15th day of application, visibly, Abgel was not only degraded but resulted in severe adhesions of internal organs and histologically capsule formation around the implant was evident. Though Surgicel was also seen as cream soft material on the site of application that joined two pieces of liver, there was no adhesion of other internal organs and histologically, immune reaction and foreign-body-type giant cells were present in large amounts. Fibrin was not found grossly on application site whereas fibrin-gelatin was seen as a small white spot. Granulation tissue formation and cell migration into the fibrin-based sheets were evident, and therefore, fibrin-based sheets are not only efficient hemostats but showed optimum degradation and wound healing.

• A freeze-dried fibrin disc as a biodegradable drug release matrix.

  Authors: T R Santhosh Kumar, Mary Vasantha Bai, Lissy K Krishnan

  Biologicals : journal of the International Association of Biological Standardization. 04/2004; 32(1):49-55.

  A fibrin clot loaded with soluble tetracycline (TET) was prepared and lyophilized to make discs of a size and shape to use as a drug delivery matrix. On subcutaneous implantation of these discs inA fibrin clot loaded with soluble tetracycline (TET) was prepared and lyophilized to make discs of a size and shape to use as a drug delivery matrix. On subcutaneous implantation of these discs in mice, they were found to have degraded in 15 days as evidenced by gross and histological examination. The in vitro discharge kinetics of tetracycline from the disc into phosphate buffered saline (PBS) and human serum were compared. It was observed that the release rate of tetracycline from the matrix into serum remained steady from day 1 to day 12, maintaining sufficient concentration that may be required to control microbial growth in the medium. Two different concentrations of fibrinogen were used to fabricate discs denoted as FG200 and FG100, and in both cases the retention rate was comparable when the study medium was serum. In contrast, when suspended in PBS instead of serum, the delivery of the drug into the medium was found to be high for up to the 3rd day when a sharp decline in discharge was observed. The fibrinogen used is a factor that determines not only the longevity of discharge but also fibrinolysis. The degradation of the disc in vitro was visible when the discs were suspended in the buffer, and correspondingly fibrin degradation product (FDP) measured in the medium using an antibody-based assay system was high. Fibrin disc is haemostatic and biodegradable in vivo, and in vitro release of a small molecule at a controlled rate is demonstrated here. Hence, it may be a suitable candidate as a drug delivery implant for short-term use.