Browsing by Author "Krishnan, LK"

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    A freeze-dried fibrin disc as a biodegradable drug release matrix
    (BIOLOGICALS, 2004)
    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 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 I 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. (C) 2004 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
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    A stable matrix for generation of tissue-engineered nonthrombogenic vascular grafts
    (TISSUE ENGINEERING, 2002)
    The potential of freeze-dried fibrin glue (FG) in combination with growth factor (GF) and gelatin (GEL) is evaluated for use as a matrix for endothelialization of artificial vascular grafts made of polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron). Improved adhesion and proliferation of human umbilical vein endothelial cells are demonstrated on different substrates coated with the FG-GF/FG-GF-GEL mixture, compared with the respective bare surfaces. The strength of adhesion of endothelial cells on the coated matrices was found to be adequate to resist shear stress when monolayers were exposed to forces of flow in an in vitro parallel plate flow chamber. The monolayers maintained physiological nonthrombogenic character as evidenced by in vitro platelet adhesion and response to agonist measurements. Nitric oxide synthesis by cells grown on the study matrices was also found to be normal. Thus, the matrix composition and the coating technique, as presented here, can be easily applied to generate tissue-engineered biomaterials with a nonthrombogenic endothelial cell monolayer for cardiovascular implants. The freeze-drying of the coated matrix ensures prolonged stability and thus the materials can be stored in a ready-to-use state for endothelial cell sodding or seeding.
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    An improved method for isolation of anti-viper venom antibodies from chicken egg yolk
    (JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, 2002)
    The production of antibodies and its purification from mammalian blood has been found low yielding and laborious. Therefore, anti snake venom antibodies for therapeutic use is obtained mostly as polyvalent whole serum or partially purified polyvalent immunoglobulin. The side effects of anti snake venom (ASV) therapy are mainly serum sickness and renal failure, which may be reduced by using sufficiently Pure antibodies. Therefore, we have standardized a simple method for production If purified antivenom. Here, we present the development of polyclonal antibodies against viper venom in liens and its isolation from the c,, yolk of immunized birds. We have modified the reported methods of purification of immunoglobulin from egg yolk, and thus yielded 90% purity of the protein. The modified method involves only two steps, such as removal of lipids from the diluted egg yolk by a freeze-thaw cycle and centrifugation, followed by gel filtration on Biogel P-150. The advantages are that the process is very simple, and from one egg, 100+/-20 mg of pure immunoglobulin is obtained. The antibodies are present in the egg for up to 100 days after the immunization. Thus, using small amounts of venom, a large quantity of the immunoglobulin is obtained in a sufficiently pure form. The antigen binding ability of the pure antibody is found good by the Ouchterlony's double diffusion experiment. (C) 22002 Published by Elsevier Science B.V.
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    Biochemical lesions of platelets stored as concentrates in PVC bags
    (INDIAN JOURNAL OF MEDICAL RESEARCH, 1997)
    Acid-base status of platelet suspension during storage is a measure of the gas permeability of the bag material. To assess the efficacy of the bags available in our market to store platelets, we compared biochemical lesions of platelets stored in an Indian polyvinyl chloride (PVC) triple bag against a Japanese PVC bag standardized for 5 days platelet storage. Platelet concentrates prepared in both control and test PVC bags were stored for 72 h. Two ml samples were drawn 1 h after preparation, and then at 24 h intervals, for analysis. Our data show that the mean pH value in the test bags was maintained above 6.5. However, the CO2 tension was high and O-2 tension was low. We also analyzed malondialdehyde (MDA) formation which is a measure of arachidonic acid metabolism, and seemed to be unaffected in stored platelets. Lactate dehydrogenase (LDH) was not released into the plasma excessively anti hence significant platelet lysis was absent during storage. Hypotonic shock response (HSR) of platelets stored in both test and control bags was comparable, indicating the possibility of satisfactory post-transfusion recovery.
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    Bioengineered fibrin-based niche to direct outgrowth of circulating progenitors into neuron-like cells for potential use in cellular therapy
    (Journal of Neural Engineering., 2015-07) Tara, S; Krishnan, LK
    Objective. Autologous cells are considered to be the best choice for use in transplantation therapy. However, the challenges and risks associated with the harvest of transplantable autologous cells limit their successful therapeutic application. The current study explores the possibility of isolating neural progenitor cells from circulating multipotent adult progenitor cells for potential use in cell-based and patient-specific therapy for neurological diseases. Approach. To enable the selection of neural progenitor cells from human peripheral blood mononuclear cells, and to support their lineage maintenance, the composition of a fibrin-based niche was optimized. Morphological examination and specific marker analysis were carried out, employing a qualitative/quantitative polymerase chain reaction followed by immunocytochemistry to: (i) characterize neural progenitor cells in culture; (ii) monitor proliferation/survival; and (iii) track their differentiation status. Main results. The presence of neural progenitors in circulation was confirmed by the presence of nestin+ cells at the commencement of the culture. The isolation, proliferation and differentiation of circulating neural progenitors to neuron-like cells were directed by the engineered niche. Neural cell isolation to near homogeneity was confirmed by the expression of β-III tubulin in ∼95% of cells, whereas microtubule associated protein-2 expression confirmed their ability to differentiate. The concentration of potassium chloride in the niche was found to favour neuron-like cell lengthening, cell-cell contact, and expressions of synaptophysin and tyrosine hydroxylase. Significance. The purpose of this research was to find out if peripheral blood could serve as a potential source of neural progenitors for cell based therapy. The study established that neural progenitors could be selectively isolated from peripheral blood mononuclear cells using a biomimetic niche. The selected cells could multiply and slowly differentiate into neuron-like cells. These neuron-like cells expressed functional proteins—tyrosine hydroxylase and synaptophysin. Early progenitors that proliferate while expressing β-III tubulin could be harvested from the culture, suggesting their potential use in cell transplantation therapy.
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    Combinatorial Application of Hyaluronic Acid and Curcumin-Albumin Conjugate for Cartilage Repair in TNF- α Induced Inflammation in Rabbit Knee Joint
    (Archives of Clinical and Biomedical Research, 2021-06) Sathee, D; Shenoy, SJ; Anil, A; Sabareeswaran, A; Krishnan, LK
    Osteoarthritis has emerged as a consequential disorder resulting from changing lifestyles, especially in the aged population. It is one of the most devastating degenerative joint diseases caused due to inflammation, wear and tear of articular cartilage leading to irreversible damage and physical trauma. Several intra-articular formulations are experimented with for restoring damaged cartilage. Many of them failed because of minimal effectiveness in establishing long-term therapeutic potential. We explored the cartilage regeneration potential of Hyaluronic acid (HA) on combining with dimethoxy curcumin-human serum albumin (DMCHSA) conjugate upon intra-articular administration. HA is known to possess immense lubrication property and is a well-recognized visco-supplement. The DMCHSA has the potential to suppress the action of inflammatory markers. So, a combinatorial approach anticipates an ideal therapeutic strategy to overcome the demerits of existing interventions. Intra-articular injection of Tumor Necrosis Factor-α (TNF-α), repeatedly at 7-day intervals disrupted the cartilage morphology and produced an inflammatory knee joint model to study the therapeutic potential of DMCHSA-HA combination. Into separate inflamed knee-joint cartilage HA, DMCHSA and DMCHSA-HA were administered periodically to highlight the advantage of mixing the latter with the former. Histopathology and gene expression analysis assessed the restoration potential of the treatment. We observed remarkable restoration of degenerated cartilage upon treatment with the DMCHSA-HA combination. The columnar arrangement of cells, regulated deposition of ECM components such as glycosaminoglycans (GAGs) & collagen, and synchronized expressions of inflammatory marker molecules suggested restoration of the treated defects. The treatments with DMCHSA, HA, or HSA alone seemed inferior to DMCHSA-HA combination therapy. The study confirmed that the combination therapy restored the damaged cartilage to normalcy.
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    Combining Biomaterials and Circulating Neural Progenitor Cells for Spinal Cord Injury Regeneration
    (TISSUE ENGINEERING PART A, 2015) Sudhadevi, T; Krishnan, LK; Vs, H; Hv, E; S, S
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    Comparative evaluation of absorbable hemostats: advantages of fibrin-based sheets
    (BIOMATERIALS, 2004)
    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 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. (C) 2004 Elsevier Ltd. All rights reserved.
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    Constitution of FibrinBased Niche for In Vitro Differentiation of Adipose-Derived Mesenchymal Stem Cells to Keratinocytes
    (BioResearch Open Access, 2015-04) Unnikrishnan, S; Jayakumar, K; Krishnan, LK
    Epithelialization of chronic cutaneous wound is troublesome and may require use of skin/cell substitutes. Adipose- derived mesenchymal stem cells (ADMSCs) have immense potential as autologous cell source for treating wounds; they can cross the germ layer boundary of differentiation and regenerate skin. When multipotent adult stem cells are considered for skin regeneration, lineage committed keratinocytes may be beneficial to prevent undesirable post-transplantation outcome. This study hypothesized that ADMSCs may be directed to epidermal lineage in vitro on a specifically designed biomimetic and biodegradable niche. Cells were seeded on the test niche constituted with fibrin, fibronectin, gelatin, hyaluronic acid, laminin V, platelet growth factor, and epidermal growth factor in the presence of cell-specific differentiation medium (DM). The ADMSCs grown on bare tissue culture polystyrene surface in DM is designated DM-control and those grown in basal medium (BM) is the BM-control. Lineage commitment was monitored with keratinocyte-specific markers such as cytokeratin 14, cytokeratin 5, cytokeratin 19, and integrin a6 at the transcriptional/translational level. The in vitro designed biomimetic fibrin composite matrix may have potential application as cell transplantation vehicle.
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    CONSTRUCTION OF SKIN TISSUE ON BIODEGRADABLE AND BIOMIMETIC SCAFFOLD USING ADIPOSE-DERIVED MESENCHYMAL STEM CELLS
    (WOUND REPAIR AND REGENERATION, 2014) Krishnan, LK; Sivan, U; Krishnan, KV
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    Contribution of fibroblasts to the mechanical stability of in vitro engineered dermal-like tissue through extra cellular matrix deposition
    (Bio Research Open Access, 2014-10) Nair, RP; Joseph, J; Harikrishnan, VS; Krishnan, VK; Krishnan, LK
    Tissue-engineered skin with mechanical and biological properties that match the native tissue could be a valuable graft to treat non-healing chronic wounds. Fibroblasts grown on a suitable biodegradable scaffold are a feasible strategy for the development of a dermal substitute above which epithelialization may occur naturally. Cell growth and phenotype maintenance are crucial to ensure the functional status of engineered tissue. In this study, an electrospun biodegradable polymer scaffold composed of a terpolymer PLGC [poly(lactide-glycolide- caprolactone)] with appropriate mechanical strength was used as a scaffold so that undesirable contraction of the wound could be prevented when it was implanted. To enhance cell growth, synthetic PLGC was incorporated with a fibrin-based biomimetic composite. The efficacy of the hybrid scaffold was evaluated by comparing it with bare PLGC in terms of fibroblast growth potential, extracellular matrix (ECM) deposition, polymer degradation, and mechanical strength. A significant increase was observed in fibroblast attachment, proliferation, and deposition of ECM proteins such as collagen and elastin in the hybrid scaffold. After growing fibroblasts for 20 d and 40 d, immunochemical staining of the decellularized scaffolds showed deposition of insoluble collagen and elastin on the hybrid scaffold but not on the bare scaffold. The loss of mechanical strength consequent to in vitro polymer degradation seemed to be balanced owing to the ECMdeposition. Thus, tensile strength and elongation were better when cells were grown on the hybrid scaffold rather than the bare samples immersed in culture medium. Similar patterns of in vivo and in vitro degradation were observed during subcutaneous implantation and fibroblast culture, respectively. We therefore postulate that a hybrid scaffold comprising PLGC and fibrin is a potential candidate for the engineering of dermal tissue to be used in the regeneration of chronic wounds.
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    Cultivation of endothelial progenitor cells on fibrin matrix and layering on dacron/polytetrafluoroethylene vascular grafts
    (ARTIFICIAL ORGANS, 2006)
    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, 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.
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    Curcumin-albumin conjugates as an effective anti-cancer agent with immunomodulatory properties
    (INTERNATIONAL IMMUNOPHARMACOLOGY, 2016) Aravind, SR; Krishnan, LK
    Curcumin (diferuloylmethane) is an active ingredient in turmeric (Curcuma longa) with anti-inflammatory, antioxidant, chemopreventive, chemosensitization, and radiosensitization properties. Conjugation of curcumin (Curc) to albumin (Alb) has been found to increase the aqueous solubility of the drug. The current study aimed to prove the safe use of the Curc-Alb conjugate in animals and to demonstrate that it retains drug action both in vitro and in vivo. Dalton's lymphoma ascites (DLA) cell viability was inhibited by the Curc-Alb conjugate in a dose dependent manner in vitro, as evidenced by the MIT assay. Administration of up to 11.4 mg of conjugated curcumin per kg body weight to healthy animals was non-toxic both in terms of lethality and weight loss. Histological analysis of vital organs (kidney, liver and spleen) also did not show toxic effects. Favorable immuno-modulatory activity was observed after continuous administration of sub-acute doses of the conjugate which caused increase in total leukocyte count, platelet count, and viable cell count in bone marrow, and enhanced proliferation of lymphocyte in vitro upon culture. In vivo studies in the DLA tumor model in mice demonstrated that conjugated drug induces tumor reduction and prevention. Significant tumor reduction was observed when the Curc-Alb conjugate was administered intraperitoneally in DLA-induced mice after 1 day (prevention therapy) and 7 days (reduction therapy) of tumor induction. There was significant reduction in both tumor volume and tumor cell numbers in the treated animals as well as a marked increase in their mean survival time and percent increase in life span. The effect was greater when the conjugate was administered soon after inducing the tumor as compared to when treatment was started after allowing tumor to grow for 7 days. Thus, the results of the present study suggest that curcumin albumin conjugate has immunomodulatory and tumor growth inhibition properties. The study postulates the drug form has the potential to be used as an anticancer agent in affected human subjects. (C) 2016 Elsevier B.V. All rights reserved.
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    Development of viper-venom antibodies in chicken egg yolk and assay of their antigen binding capacity
    (TOXICON, 2002) Devi, CM; Bai, MV; Krishnan, LK
    The therapeutic use of specific antibodies is invaluable in certain clinical conditions, such as administration of specific antivenom for snakebite envenomation. The production of antibodies and their purification from mammalian blood has been found low yielding and laborious. Most antivenom is polyvalent whole serum or partially purified immunoglobulin. The side effects of anti-snake-venom therapy include serum sickness and can be reduced by using mono-specific antivenom in sufficiently pure form. We have attempted to standardize a simple method for producing avian antivenom in relatively pure form from eggs. The isolation is very simple and involves only two steps, namely, removal of lipids from the diluted egg yolk followed by gel filtration. Each egg produces 80-100 mg of pure immunoglobulin, and specific antibodies are present for up to 100 days after immunization. Thus, large quantities of the Ig can be obtained in pure form using only small amounts of venom. Antigen binding was shown by Ouchterlony's double diffusion experiments and the avian antivenom neutralizes the thrombin-like activity of equivalent amounts of venom on human plasma. The LD50 of the venom was similar to3 mg/kg body weight in mice and rats but when pre-incubated with equivalent amounts (by weight) of egg IgG injected subcutaneously, all the animals survived. In a similar experiment using a commercial horse IgG, 25% mortality is seen. These results indicate that the antivenom immunoglobulins purified from immunized chicken egg yolk is biologically active and the possibility of their therapeutic use will be investigated further. (C) 2002 Elsevier Science Ltd. All rights reserved.
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    Effect of matrix composition on differentiation of nestin-positive neural progenitors from circulation into neurons
    (JOURNAL OF NEURAL ENGINEERING, 2010) Jose, A; Krishnan, LK
    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 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.
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    Effect of passage number and matrix characteristics on differentiation of endothelial cells cultured for tissue engineering
    (BIOMATERIALS, 2005) Chennazhy, KP; Krishnan, LK
    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 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 Lire 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 Lire 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. (c) 2005 Published by Elsevier Ltd.
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    Effect of temperature on functional response of platelet concentrates stored in PVC bags
    (INDIAN JOURNAL OF MEDICAL RESEARCH, 1997)
    Haemostatic efficacy of platelet concentrates prepared and stored in locally available PVC triple bags was compared against a Japanese bag. In vitro functional parameters studied included shape change, aggregation and secretion in response to ADP. We have observed remarkable difference in the aggregatory response of platelets stored at slightly varying temperatures. The stimulatory responses of platelets stored with constant agitation at 70 strokes per min and 23+/-2 degrees C, deteriorated drastically by the time platelets were stored for 48 h. Both the rate and the extent of aggregation were affected showing no response to ADP at 72 h. However, when platelets were stored in a BOD incubator, thermostated at 22+/-0.5 degrees C, with continuous horizontal agitation at 70 strokes per min, 50 per cent functional response was retained till 72 h. We also demonstrated fragmentation of platelet membrane during storage. The membrane fragments collected bq. high speed centrifugation, expressed PF3 activity. Shedding of microvesicles indicates alterations at the membrane level that possibly cause functional lesion during storage. Our data suggest the significance of controlling the storage temperature steadily, to get maximum post transfusion efficacy.
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    Endothelial cell growth factor (ECGF) enmeshed with fibrin matrix enhances proliferation of EC in vitro
    (BIOMATERIALS, 2001) Kumar, TRS; Krishnan, LK
    The vascular biomaterials that are currently used for clinical implants have been considered as poor substrates for human endothelial cell adhesion and spreading. Therefore, thrombotic occlusion is the predominant cause for the failure of small diameter vascular grafts made out of Dacron or Teflon. To reduce surface thrombogenicity of material surfaces used for vascular implants, in vitro seeding of endothelial cells using adhesive protein matrix is under evaluation in various laboratories. Evidences suggest that fibrin matrix is a suitable matrix for endothelial cell (EC) adhesion to the currently available vascular graft materials; however, poor proliferation of attached cells seems to be a major limitation. During this study we have also found that fibrin is a better matrix compared to gelatin to support cell attachment and spreading. However, the poor proliferation of initially attached human umbilical cord vein endothelial cell (HUVEC) necessitated modification of the matrix composition to get a monolayer within a limited period. Since fibrin can form a network of protein bundles, an effort is made to incorporate growth factors within the matrix. Endothelial cell growth factor (ECGF) isolated from bovine hypothalamus is immobilized on the surface with fibrin glue (FG) to promote proliferation of HUVEC. The results demonstrate that proteins with similar molecular weights as growth factors (GF) are retained within the matrix and released into the culture medium for 96 h, in quantities that would be sufficient to promote cell proliferation. When cells were seeded on the matrix composed with components of FG and ECGF, the HUVEC proliferated at a significantly higher rate compared to the cells on surfaces coated with gelatin or fibrin. The EC thus grown on the composite (FG + ECGF) resisted the shear stress as compared to the cells grown on gelatin. The HUVEC monolayer grown on the composite seems thromboresistant as adhesion and activation of platelets are negligible after platelet rich plasma is incubated with the monolayer for about 1 h with agitation. Therefore, the composite of fibrin and ECGF can be a suitable matrix for further evaluation of patients' autologous endothelial cell attachment and proliferation for clinical application. (C) 2001 Elsevier Science Ltd. All rights reserved.