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Sample records for platelet lysate preparation

  1. Comparative analyses of industrial-scale human platelet lysate preparations.

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    Pierce, Jan; Benedetti, Eric; Preslar, Amber; Jacobson, Pam; Jin, Ping; Stroncek, David F; Reems, Jo-Anna

    2017-12-01

    Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product. Nine industrial-scale, serum-based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet-rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma-based or serum-based PL were compared to MSCs cultured with fetal bovine serum. Electrolyte and protein levels were relatively consistent among all serum-based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum-based PL stored at -80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum-based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement. Using a large-scale, standardized method, lot-to-lot variations were noted for industrial-scale preparations of serum-based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off-the-shelf PL is a feasible substitute for fetal bovine serum in MSC cultures. © 2017 AABB.

  2. Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study

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    Franka Klatte-Schulz

    2018-01-01

    Full Text Available The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP; RegenLab (PRP-BCT, platelet concentrate (apheresis, PC, and PL (freezing-thawing destruction of PC. Additionally, ten commercial allogenic PLs (AlloPL from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

  3. Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations.

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    Boraldi, Federica; Burns, Jorge S; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela

    2018-03-01

    Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow-derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective

  4. Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.

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    Sandra Mjoll Jonsdottir-Buch

    Full Text Available BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. CONCLUSION/SIGNIFICANCE: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

  5. Platelet lysate embedded scaffolds for skin regeneration.

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    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Cervio, Marila; Riva, Federica; Liakos, Ioannis; Athanassiou, Athanassia; Saporito, Francesca; Marini, Lara; Caramella, Carla

    2015-04-01

    The work presents the development of acellular scaffolds extemporaneously embedded with platelet lysate (PL), as an innovative approach in the field of tissue regeneration/reparation. PL embedded scaffolds should have a tridimensional architecture to support cell migration and growth, in order to restore skin integrity. For this reason, chondroitin sulfate (CS) was associated with sodium alginate (SA) to prepare highly porous systems. The developed scaffolds were characterized for chemical stability to γ-radiation, morphology, hydration and mechanical properties. Moreover, the capability of fibroblasts and endothelial cells to populate the scaffold was evaluated by means of proliferation test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and confocal laser scanning microscopy study. The scaffolds, not altered by sterilization, were characterized by limited swelling and high flexibility, by foam-like structure with bubbles that formed a high surface area and irregular texture suitable for cell adhesion. Cell growth and scaffold population were evident on the bubble surface, where the cells appeared anchored to the scaffold structure. Scaffold network based on CS and SA demonstrated to be an effective support to enhance and to allow fibroblasts and endothelial cells (human umbilical vein endothelial cells, HUVEC) adhesion and proliferation. In particular, it could be hypothesized that cell adhesion was facilitated by the synergic effect of PL and CS. Although further in vivo evaluation is needed, on the basis of in vitro results, PL embedded scaffolds seem promising systems for skin wound healing.

  6. Porcine platelet lysate as a supplement for animal cell culture

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    Aldén, Anna; Gonzalez, Lorena; Persson, Anna; Christensson, Kerstin; Holmqvist, Olov

    2007-01-01

    A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products. PMID:19002989

  7. Analysis of Reparative Activity of Platelet Lysate: Effect on Cell Monolayer Recovery In Vitro and Skin Wound Healing In Vivo.

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    Sergeeva, N S; Shanskii, Ya D; Sviridova, I K; Karalkin, P A; Kirsanova, V A; Akhmedova, S A; Kaprin, A D

    2016-11-01

    Platelet lysate prepared from donor platelet concentrate and pooled according to a developed technique stimulates migration of multipotent mesenchymal stromal cells of the human adipose tissue and promotes healing of the monolayer defect in cultures of human fibroblasts and multipotent mesenchymal stromal cells in vitro in concentrations close those of fetal calf serum (5-10%). Lysate of platelets from platelet-rich rat blood plasma stimulated healing of the skin defect by promoting epithelialization and granulation tissue formation. The regenerative properties of platelet lysate in vivo increased with increasing its concentration.

  8. Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

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    Lykov, A P; Bondarenko, N A; Surovtseva, M A; Kim, I I; Poveshchenko, O V; Pokushalov, E A; Konenkov, V I

    2017-10-01

    We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

  9. Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

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    Johansson, Liselott; Klinth, Jeanna; Holmqvist, Olov; Ohlson, Sten

    2003-01-01

    A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture stud...

  10. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

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    Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

    2016-01-01

    The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors.

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    Sumner, Scarlett M; Naskou, Maria C; Thoresen, Merrilee; Copland, Ian; Peroni, John F

    2017-07-01

    Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and coagulation panels and then again at 8, 16, 24, and 48 hours after the procedure. To produce platelet lysate, the platelet concentrate underwent two freeze-thaw cycles followed by centrifugation and filtration processing. The platelet lysate was then analyzed for cellular debris, fibrinogen, and growth factors. The collected platelet concentration contained a mean platelet yield of 390 × 10 3 /μL. Donor platelet count decreased from a mean of 193 × 10 3 /μL to 138 × 10 3 /μL after plateletpheresis, but no individual was at risk for hemorrhage. Pooled platelet lysate had minimal cellular residue and contained growth factor concentrations at 6.1 ng/mL for transforming growth factor-β1, at 3.5 ng/mL for platelet-derived growth factor-BB, and at 13.8 ng/mL for vascular endothelial growth factor-A. Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture. © 2017 AABB.

  12. Platelet lysate-based pro-angiogenic nanocoatings.

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    Oliveira, Sara M; Pirraco, Rogério P; Marques, Alexandra P; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

    2016-03-01

    Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20 h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenic-associated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. The presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  13. Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers.

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    Sovkova, Vera; Vocetkova, Karolina; Rampichova, Michala; Mickova, Andrea; Buzgo, Matej; Lukasova, Vera; Dankova, Jana; Filova, Eva; Necas, Alois; Amler, Evzen

    2018-06-01

    Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

  14. Exosomes: novel effectors of human platelet lysate activity

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    E Torreggiani

    2014-09-01

    Full Text Available Despite the popularity of platelet-rich plasma (PRP and platelet lysate (PL in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF, vascular endothelial growth factor (VEGF, platelet-derived growth factor (PDGF-BB and transforming growth factor beta 1 (TGF-β1 as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

  15. Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

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    Yvonne Wanner

    2017-01-01

    Full Text Available Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL- cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

  16. Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization.

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    Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar; Alexander, Dorothea

    2017-01-01

    Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

  17. Effect of platelet lysate on human cells involved in different phases of wound healing.

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    Maria Chiara Barsotti

    Full Text Available BACKGROUND: Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization. METHODOLOGY/PRINCIPAL FINDINGS: Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2 and inflammatory response evaluation (NFκB. Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v. Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control, comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. CONCLUSION/SIGNIFICANCE: These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  18. Effect of platelet lysate on human cells involved in different phases of wound healing.

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    Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

    2013-01-01

    Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (pplatelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  19. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

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    Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

    2016-05-04

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

  20. The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

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    Moreira Teixeira, Liliana S; Leijten, Jeroen C H; Wennink, Jos W H; Chatterjea, Anindita G; Feijen, Jan; van Blitterswijk, Clemens A; Dijkstra, Pieter J; Karperien, Marcel

    2012-05-01

    In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat-derived platelet concentrates.

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    Glovinski, Peter V; Herly, Mikkel; Mathiasen, Anders B; Svalgaard, Jesper D; Borup, Rehannah; Talman, Maj-Lis M; Elberg, Jens J; Kølle, Stig-Frederik T; Drzewiecki, Krzysztof T; Fischer-Nielsen, Anne

    2017-02-01

    Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights

  2. Platelet lysate formulations based on mucoadhesive polymers for the treatment of corneal lesions.

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    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Del Fante, Claudia; Perotti, Cesare; Scudeller, Luigia; Caramella, Carla

    2011-02-01

    Growth factors contained in platelet α-granules initiate and modulate tissue repair and are proposed for the treatment of soft and hard-tissue surgical conditions and in the management of non-healing wounds. Platelet lysate is a hemoderivative obtained from platelet-rich plasma and is capable of releasing a pool of growth factors. Many medical and surgical techniques have been proposed for the treatment of corneal lesions; management of these conditions remains problematic and healing with standard protocols is unattainable. The aim of this study was to develop formulations suitable for prolonging the contact of platelet lysate with the damaged cornea for the time necessary to exert a therapeutic effect. Two vehicles, one based on polyacrylic acid and one based on chitosan, were autoclaved and loaded with platelet lysate and the resultant formulations were characterized for rheology, mucoadhesion, vehicle compatibility and stability. The proliferation effect was tested on two cell culture types (rabbit corneal epithelial cells and fibroblasts). An in-vitro wound-healing test was performed on fibroblasts. In both cases the formulations were compared with platelet lysate diluted with saline at the same concentration. Both formulations maintained the rheological and mucoadhesive properties of the vehicles and the proliferative activity of platelet lysate. The chitosan formulation was able to significantly enhance epithelial cell growth even after storage of up to 2 weeks (in-use conditions), while the polyacrylic acid formulation was less efficient, probably due to the characteristics of the cell model used. The in-vitro wound-healing test performed on fibroblasts confirmed the differences between the two vehicles. The effect induced by the platelet lysate and chitosan formulation was faster than that of the polyacrylic acid formulation and complete in-vitro wound repair was achieved within 48 h. © 2010 The Authors. JPP © 2010 Royal Pharmaceutical Society.

  3. Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

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    Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

    2013-11-01

    We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum.

  4. Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

    Science.gov (United States)

    Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

    2016-10-01

    To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

  5. Wound Healing Activity of a New Formulation from Platelet Lysate

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    Akram Jamshidzadeh

    2016-03-01

    Full Text Available Platelet-rich plasma (PRP is an attractive preparation in regenerative medicine due to its potential role in the healing process in different experimental models. This study was designed to investigate the wound healing activity of a new formulation of PRP. Different gel-based formulations of PRP were prepared. Open excision wounds were made on the back of male Sprague-Dawley rats, and PRP gel was administered topically once daily until the wounds healed completely (12 days. The results revealed that the tested PRP formulation significantly accelerated the wound healing process by increasing the wound contraction, tissue granulization, vascularization, and collagen regeneration. Interestingly, this study showed that there were no significant differences between the PRP and its gel-based formulation in all the above mentioned parameters. Although this investigation showed that PRP formulation had significant wound healing effects, the PRP gel-based formulation also had significant wound healing properties. This might indicate the wound healing properties of the PRP gel ingredients in the current investigation.

  6. Electrospun Gelatin–Chondroitin Sulfate Scaffolds Loaded with Platelet Lysate Promote Immature Cardiomyocyte Proliferation

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    Francesca Saporito

    2018-02-01

    Full Text Available The aim of the present work was the development of heart patches based on gelatin (G and chondroitin sulfate (CS to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD. Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system’s general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

  7. The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation

    NARCIS (Netherlands)

    Moreira Teixeira, Liliana; Leijten, Jeroen Christianus Hermanus; Wennink, J.W.H.; Ganguly, Anindita; Feijen, Jan; van Blitterswijk, Clemens; Dijkstra, Pieter J.; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and

  8. Platelet lysate as an autologous alternative for fetal bovine serum in cardiovascular tissue engineering

    NARCIS (Netherlands)

    Riem Vis, P.W.; Bouten, C.V.C.; Sluijter, J.P.G.; Herwerden, van L.A.; Kluin, J.

    2010-01-01

    There is an ongoing search for alternative tissue culture sera to engineer autologous tissues, since use of fetal bovine serum (FBS) is limited under Good Tissue Practice (GTP) guidelines. We compared FBS with human Platelet-lysate (PL) in media for in vitro cell culture. A threefold increase in

  9. Platelet lysate and chondroitin sulfate loaded contact lenses to heal corneal lesions.

    Science.gov (United States)

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Delfino, Alessio; Riva, Federica; Icaro Cornaglia, Antonia; Marrubini, Giorgio; Musitelli, Giorgio; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

    2016-07-25

    Hemoderivative tear substitutes contain various ephiteliotrophic factors, such as growth factors (GF), involved in ocular surface homeostasis without immunogenic properties. The aim of the present work was the loading of platelet lysate into contact lenses to improve the precorneal permanence of platelet lysate growth factors on the ocular surface to enhance the treatment of corneal lesions. To this purpose, chondroitin sulfate, a sulfated glycosaminoglycan, which is normally present in the extracellular matrix, was associated with platelet lysate. In fact, chondroitin sulfate is capable of electrostatic interaction with positively charged growth factors, in particular, with bFGF, IGF, VEGF, PDGF and TGF-β, resulting in their stabilization and reduced degradation in solution. In the present work, various types of commercially available contact lenses have been loaded with chondroitin sulfate or chondroitin sulfate in association with platelet lysate to achieve a release of growth factors directly onto the corneal surface lesions. One type of contact lenses (PureVision(®)) showed in vitro good proliferation properties towards corneal cells and were able to enhance cut closure in cornea constructs. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    Science.gov (United States)

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

    Science.gov (United States)

    Iudicone, Paola; Fioravanti, Daniela; Bonanno, Giuseppina; Miceli, Michelina; Lavorino, Claudio; Totta, Pierangela; Frati, Luigi; Nuti, Marianna; Pierelli, Luca

    2014-01-27

    Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic

  12. Use of platelet lysate for bone regeneration - are we ready for clinical translation?

    Science.gov (United States)

    Altaie, Ala; Owston, Heather; Jones, Elena

    2016-02-26

    Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its "potency" assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

  13. GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

    Science.gov (United States)

    Carducci, Augusto; Scafetta, Gaia; Siciliano, Camilla; Carnevale, Roberto; Rosa, Paolo; Coccia, Andrea; Mangino, Giorgio; Bordin, Antonella; Vingolo, Enzo Maria; Pierelli, Luca; Lendaro, Eugenio; Ragona, Giuseppe; Frati, Giacomo; De Falco, Elena

    2016-01-01

    Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

  14. Tailor-made purified human platelet lysate concentrated in neurotrophins for treatment of Parkinson's disease.

    Science.gov (United States)

    Chou, Ming-Li; Wu, Joe-Wei; Gouel, Flore; Jonneaux, Aurélie; Timmerman, Kelly; Renn, Ting-Yi; Laloux, Charlotte; Chang, Hung-Ming; Lin, Liang-Tzung; Devedjian, Jean-Christophe; Devos, David; Burnouf, Thierry

    2017-10-01

    Human platelet lysates (PLs), which contain multiple neurotrophins, have been proposed for treating neurodegenerative disorders, including Parkinson's disease (PD). However, current PLs suspended in plasma have high protein content and contain fibrinogen/fibrin and, following activation, also proteolytic and thrombogenic enzymes. Upon brain administration, such PLs may saturate the cerebrospinal fluid and exert neurotoxicity. We assessed whether purified PLs, concentrated in neurotrophins, protected dopaminergic neurons in PD models. Platelet concentrates were collected by apheresis and centrifuged to eliminate plasma and recover the platelets. Platelets were lysed by freeze-thaw cycles, and the 10-fold concentrated platelet pellet lysates (PPLs) were heat-treated (at 56 °C for 30 min). The heat-treated PPLs were low in total proteins, depleted in both plasma and platelet fibrinogen, and devoid of thrombogenic and proteolytic activities. They exerted very high neuroprotective activity when non-oncogenic, Lund human mesencephalic (LUHMES) cells that had differentiated into dopaminergic neurons were exposed to the MPP + neurotoxin. Heat treatment improved the neuroprotection and inactivated the neurotoxic blood-borne hepatitis C virus. PPL did not induce inflammation in BV2 microglial cells and inhibited COX-2 expression upon lipopolysaccharide exposure. Intranasal administration in mice revealed (a) diffusion of neurotrophins in the striatum and cortex, and (b) MPTP intoxication neuroprotection in the substantia nigra and striatum and the absence of neuroinflammation. These dedicated heat-treated PPLs can be a safe and valuable candidate for a therapeutic strategy for PD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Development and validation of a production process of platelet lysate for autologous use.

    Science.gov (United States)

    Plöderl, Karin; Strasser, Cornelia; Hennerbichler, Simone; Peterbauer-Scherb, Anja; Gabriel, Christian

    2011-01-01

    Growth factors (GF) contained in platelets are a potential source to improve wound healing by the stimulation and acceleration of soft tissue and bone healing. This resulted in the idea that autologous platelet-rich plasma or platelet lysate (PL) containing high levels of GF might improve healing processes. Today platelet products are already applied in bone and maxillofacial surgery. In recent years, cosmetic surgery and facial rejuvenation procedures are growing steadily. New methods including platelet products aiming to induce non-surgical reduction of wrinkles upon topical injection and to minimize surgical risks in general are developed. Several point-of-care devices are already available on the market. However, the amount of PL obtained by these kits is far too high for certain applications in cosmetic surgery and they offer no possibility of storing the remaining material in a sterile manner. Therefore we developed a procedure for the sterile production of smaller amounts of PL in a closed system that can also be split into several products for repeated administration. The closed system was determined to be a bag system designed for an autologous blood donation of 100 ml whole blood. We set a special focus on the validation of the production procedure, mainly regarding sterility and platelet recovery. For validation 22 healthy volunteers were asked for a blood donation, which was centrifuged twice to obtain concentrated platelets (CP). A freeze-thaw cycle caused lysis of the CP to get approximately 8.48 ± 1.36 ml PL. We yielded satisfying results of 100% sterility and a platelet recovery of 36.92% ± 18.71%. We therefore conclude that the PL obtained is ready for studies comparing it with traditional treatments.

  16. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

    Directory of Open Access Journals (Sweden)

    Antonio M. Fea

    2016-01-01

    Full Text Available Purpose. To determine the effectiveness of autologous platelet lysate (APL eye drops in patients with primary Sjögren syndrome (SS dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM. Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI, best corrected visual acuity (BCVA, Schirmer test, fluorescein score, and breakup time (BUT. A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n=20 patients mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n=10 patients. The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p<0.05. Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

  17. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study.

    Science.gov (United States)

    Fea, Antonio M; Aragno, Vittoria; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

  18. Virally inactivated human platelet concentrate lysate induces regulatory T cells and immunosuppressive effect in a murine asthma model.

    Science.gov (United States)

    Lee, Yueh-Lun; Lee, Lin-Wen; Su, Chen-Yao; Hsiao, George; Yang, Yi-Yuan; Leu, Sy-Jye; Shieh, Ying-Hua; Burnouf, Thierry

    2013-09-01

    Platelet concentrate lysates (PCLs) are increasingly used in regenerative medicine. We have developed a solvent/detergent (S/D)-treated PCL. The functional properties of this preparation should be unveiled. We hypothesized that, due to transforming growth factor-β1 (TGF-β1) content, PCLs may exert immunosuppressive and anti-inflammatory functions. PCL was prepared by S/D treatment, oil extraction, and hydrophobic interaction chromatography. The content of TGF-β in PCL was determined by enzyme-linked immunosorbent assay. Cultured CD4+ T cells were used to investigate the effects of PCL on expression of transcription factor forkhead box P3 (Foxp3), the inhibition of T-cell proliferation, and cytokine production. The regulatory function of PCL-converted CD4+ T cells was analyzed by suppressive assay. The BALB/c mice were given PCL-converted CD4+ T cells before ovalbumin (OVA) sensitization and challenge using an asthma model. Inflammatory parameters, such as the level of immunoglobulin E (IgE), airway hyperresponsiveness (AHR), bronchial lavage fluid eosinophils, and cytokines were assayed. Recombinant human (rHu) TGF-β1 was used as control. PCL significantly enhanced the development of CD4+Foxp3+-induced regulatory T cells (iTregs). Converted iTregs produced neither Th1 nor Th2 cytokines and inhibited normal T-cell proliferation. PCL- and rHuTGF-β-converted CD4+ T cells prevented OVA-induced asthma. PCL- and rHuTGF-β-modified T cells both significantly reduced expression levels of OVA-specific IgE and significantly inhibited the development of AHR, airway eosinophilia, and Th2 responses in mice. S/D-treated PCL promotes Foxp3+ iTregs and exerts immunosuppressive and anti-inflammatory properties. This finding may help to understand the clinical properties of platelet lysates. © 2013 American Association of Blood Banks.

  19. Platelet lysate mucohadesive formulation to treat oral mucositis in graft versus host disease patients: a new therapeutic approach.

    Science.gov (United States)

    Del Fante, Claudia; Perotti, Cesare; Bonferoni, Maria Cristina; Rossi, Silvia; Sandri, Giuseppina; Ferrari, Franca; Scudeller, Luigia; Caramella, Carla Marcella

    2011-09-01

    Optimal treatment of oral mucositis (OM) due to graft versus host disease (GvHD) is currently not available. Platelet-derived growth factors (PDGFs) have high capability for tissue healing and may play a role in repairing the mucosal barrier. The aim of the present work was to develop a mucoadhesive formulation to administer platelet lysate to oral cavity prolonging contact time of platelet lysate with oral mucosa. The mucoadhesive formulation was characterized for in vitro properties (PDGF-AB concentration, mucoadhesive properties, cytotoxicity, fibroblast proliferation, wound healing). Moreover, a preliminary clinical study on seven GvHD patients with OM refractory to other therapies was conducted, to evaluate feasibility, safety, and efficacy. GVPL (mucoadhesive gel vehicle mixed with platelet lysate)showed good mucoadhesive properties; additionally, it was characterized by good biocompatibility in vitro on fibroblasts and it was able to enhance fibroblast proliferation and wound healing, maintaining the efficacy for up to 14 days following storage at 2-8°C. In vivo, clinical response was good-to-complete in five, fair in one, none in the remaining one. The in vitro results indicate that GVPL has optimal mucoadhesive and healing enhancer properties, maintained over time (up to 14 days); preliminary clinical results suggest that oral application of platelet lysate-loaded mucoadhesive formulation is feasible, safe, well tolerated, and effective. A larger controlled randomized study is needed.

  20. Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

    Science.gov (United States)

    Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-03-01

    Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

  1. Topical gel formulation and stability assessment of platelet lysate based on turbidimetric method

    Directory of Open Access Journals (Sweden)

    Soliman Mohammadi Samani

    2015-06-01

    Full Text Available Platelet-rich growth factors have attracted attentions of scientists and clinical practitioners who are involved in wound healing and regenerative medicine extensively, according to their unprecedented potential of promoting and catalyzing healing process. Platelet-rich growth factors are cost-benefit, available and more stable than recombinant human growth factors. These appealing characteristics have converted PRGF to one of the popular candidates for treatment of variety of wounds. According to these valuable properties, we decided to formulate and assess the effect of different excipients on the stability of such valuable protein based formulations. Different excipients have been chosen according to their effective ness on the stability of proteins and their application in other similar formulations. The stabilizing effect of excipients was evaluated by measuring heat-induced aggregation of growth factors by turbidimetric assay. Glycerol, glycine and dextrose were chosen as stabilizing excipients for these formulations. The results show that dextrose has more stabilizing effect on prevention of heat induced aggregation of the platelet lysate growth factors than glycerol and glycine. All of the formulations also contained antioxidant, chelating agents, preservative and carbopol934 in order to form appropriate gel.

  2. Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units.

    Science.gov (United States)

    Sellberg, Felix; Berglund, Erik; Ronaghi, Martin; Strandberg, Gabriel; Löf, Helena; Sommar, Pehr; Lubenow, Norbert; Knutson, Folke; Berglund, David

    2016-12-01

    Platelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units. Platelet pooled units (n = 10) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-β isoform 1, IL-1β, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ. The concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-β, PDGF-AB/BB, and PF4 showed an increase of 27.2% (p product, which potentially may influence the clinical effects. Copyright © 2016. Published by Elsevier Ltd.

  3. Evaluation of a platelet lysate bilayered system for periodontal regeneration in a rat intrabony three-wall periodontal defect

    NARCIS (Netherlands)

    Babo, P.S.; Cai, X; Plachokova, A.S.; Reis, R.L.; Jansen, J.A.; Gomes, M.E.; Walboomers, X.F.

    2018-01-01

    With currently available therapies, full regeneration of lost periodontal tissues after periodontitis cannot be achieved. In this study, a combined compartmentalized system was tested, composed of (a) a platelet lysate (PL)-based construct, which was placed along the root aiming to regenerate the

  4. Decreased mechanical properties of heart valve tissue constructs cultured in platelet lysate as compared to fetal bovine serum

    NARCIS (Netherlands)

    Geemen, van D.; Riem Vis, P.W.; Soekhradj - Soechit, R.S.; Sluijter, J.P.G.; Liefde - van Beest, de M.; Kluin, J.; Bouten, C.V.C.

    2011-01-01

    In autologous heart valve tissue engineering, there is an ongoing search for alternatives of fetal bovine serum (FBS). Human platelet-lysate (PL) might be a promising substitute. In the present article, we aimed to examine the tissue formation, functionality, and mechanical properties of engineered

  5. Autologous platelet lysate local injections for the treatment of refractory lateral epicondylitis.

    Science.gov (United States)

    Tan, Xun-xiang; Ju, Hai-yang; Yan, Wei; Jiang, Hong-jiang; Su, Jin-ping; Dong, Hua-jun; Wang, Ling-shuang; Zou, De-bao

    2016-01-25

    The purpose of this study was to investigate the efficacy and safety of autologous platelet lysate (APL) local injections in reducing pain and improving function in patients with refractory lateral epicondylitis. A total of 56 patients with refractory lateral epicondylitis were enrolled in this study. All the patients received three injections in one course of treatment. Subjective assessments include visual analog scale (VAS) pain score and Mayo elbow score before injection (baseline) and at 1, 6, and 12 months after injection. Significant differences were observed in VAS and Mayo scores at baseline and at 1, 6, and 12 months after injection. Overall, the injections of APL improved local symptoms and all the patients recovered to normal elbow function with 12 months follow-up. Local injections of APL resulted in favorable clinical outcomes for the treatment of lateral epicondylitis. APL could be clinically effective in the treatment of lateral epicondylitis.

  6. Assessment of bone healing ability of calcium phosphate cements loaded with platelet lysate in rat calvarial defects.

    Science.gov (United States)

    Babo, Pedro S; Carvalho, Pedro P; Santo, Vítor E; Faria, Susana; Gomes, Manuela E; Reis, Rui L

    2016-11-01

    Injectable calcium phosphate cements have been used as a valid alternative to autologous bone grafts for bone augmentation with the additional advantage of enabling minimally invasive implantation procedures and for perfectly fitting the tissue defect. Nevertheless, they have low biodegradability and lack adequate biochemical signaling to promote bone healing and remodeling. In previous in vitro studies, we observed that the incorporation of platelet lysate directly into the cement paste or loaded in hyaluronic acid microspheres allowed to modulate the cement degradation and the in vitro expression of osteogenic markers in seeded human adipose derived stem cells. The present study aimed at investigating the possible effect of this system in new bone formation when implanted in calvarial bilateral defects in rats. Different formulations were assessed, namely plain calcium phosphate cements, calcium phosphate cements loaded with human platelet lysate, hybrid injectable formulations composed of the calcium phosphate cement incorporating hyaluronin acid non-loaded microparticles (20% hyaluronin acid) or with particles loaded with platelet lysate. The degradability and new bone regrowth were evaluated in terms of mineral volume in the defect, measured by micro-computed tomography and histomorphometric analysis upon 4, 8 and 12 weeks of implantation. We observed that the incorporation of hyaluronin acid microspheres induced an overly rapid cement degradation, impairing the osteoconductive properties of the cement composites. Moreover, the incorporation of platelet lysate induced higher bone healing than the materials without platelet lysate, up to four weeks after surgery. Nevertheless, this effect was not found to be significant when compared to the one observed in the sham-treated group. © The Author(s) 2016.

  7. Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

    2017-10-01

    The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy. Copyright© by Royan Institute. All rights reserved.

  8. Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate.

    Science.gov (United States)

    Oliveira, Sara M; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

    2015-04-01

    Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P > 5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

  10. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

    Science.gov (United States)

    Bieback, Karen

    2013-10-01

    Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.

  11. Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

    Science.gov (United States)

    Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

    2016-01-13

    The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds.

  12. The effects of platelet lysate patches on the activity of tendon-derived cells.

    Science.gov (United States)

    Costa-Almeida, Raquel; Franco, Albina R; Pesqueira, Tamagno; Oliveira, Mariana B; Babo, Pedro S; Leonor, Isabel B; Mano, João F; Reis, Rui L; Gomes, Manuela E

    2018-03-01

    Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards

  13. Calcium alginate particles for the combined delivery of platelet lysate and vancomycin hydrochloride in chronic skin ulcers.

    Science.gov (United States)

    Mori, Michela; Rossi, Silvia; Bonferoni, Maria Cristina; Ferrari, Franca; Sandri, Giuseppina; Riva, Federica; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla

    2014-01-30

    The aim of the present work was the development of a powder formulation for the combined delivery of platelet lysate and of a model antibiotic drug, vancomycin hydrochloride (VCM), in chronic skin ulcers. Calcium alginate particles were prepared by freeze-drying beads obtained by ionic gelation method. The experimental conditions adopted permitted the complete loading of VCM and of PDGF AB, the growth factor chosen as representative of those contained in PL. Such particles where able to absorb PBS (mimicking wound exudate), to form a gel and to modulate the release of VCM and of PDGF AB. They are characterized by enhancement properties of human fibroblast proliferation due to PL presence. In particular, PL, when loaded in alginate particles, was able not only to increase the number of viable cells, but also the number of cells in proliferative phase. Such properties were comparable to those of fresh PL indicating the capability of calcium alginate particles to load PL bioactive substances without altering their activity. The formulation developed is characterized by an easier and a less painful administration with respect to traditional gauzes and semisolid preparations and permits the loading in the same dosage form of active substances of different nature avoiding eventual incompatibility problems. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

    Science.gov (United States)

    Robinson, Scott T; Douglas, Alison M; Chadid, Tatiana; Kuo, Katie; Rajabalan, Ajai; Li, Haiyan; Copland, Ian B; Barker, Thomas H; Galipeau, Jacques; Brewster, Luke P

    2016-05-01

    Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL

  15. Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.

    Science.gov (United States)

    Pignatelli, Cataldo; Perotto, Giovanni; Nardini, Marta; Cancedda, Ranieri; Mastrogiacomo, Maddalena; Athanassiou, Athanassia

    2018-04-17

    Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and

  16. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Michael Lohmann

    Full Text Available The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS or other serum supplements such as human platelet lysate (HPL. In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1, but it was significantly higher with HPLs from younger donors (45 years. Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal. HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1 or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3 were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

  17. Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use.

    Science.gov (United States)

    Altaie, Ala; Baboolal, Thomas G; Wall, Owen; Jones, Elena; McGonagle, Dennis

    2018-03-01

    Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid-derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit-fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca ++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

    Science.gov (United States)

    Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

    2012-01-01

    The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation. PMID:22662236

  19. Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds.

    Science.gov (United States)

    Bonferoni, Maria Cristina; Sandri, Giuseppina; Rossi, Silvia; Dellera, Eleonora; Invernizzi, Alessandro; Boselli, Cinzia; Cornaglia, Antonia Icaro; Del Fante, Claudia; Perotti, Cesare; Vigani, Barbara; Riva, Federica; Caramella, Carla; Ferrari, Franca

    2018-02-09

    Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

  20. Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds

    Directory of Open Access Journals (Sweden)

    Maria Cristina Bonferoni

    2018-02-01

    Full Text Available Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

  1. Evaluation of intra-articular injection of autologous platelet lysate (PL) in horses with osteoarthritis of the distal interphalangeal joint.

    Science.gov (United States)

    Tyrnenopoulou, Panagiota; Diakakis, Nikolaos; Karayannopoulou, Maria; Savvas, Ioannis; Koliakos, Georgios

    2016-06-01

    Regenerative medicine has become one of the most promising therapies of equine osteoarthritis. Platelet lysate (PL) is rich in bioactive proteins and growth factors that play a crucial role in tissue healing. To evaluate the efficacy of intra-articularly injected autologous PL in equine athletes with naturally occurring osteoarthritis. Fifteen warmblood geldings aged 8-19 years with osteoarthritis of the distal interphalangeal joint were included in this study. They were randomly divided into two groups; 10 horses received intra-articular injections of PL and 5 of normal saline (controls). Before treatment, platelet-derived growth factor (PDGF) levels in basal plasma and prepared PL were estimated. Each joint was injected twice within a three-week period. Lameness was evaluated using the American Association of Equine Practitioners grading system, before treatment and 10 days after each intra-articular injection. Horses were examined fortnightly for one year. Radiographic examination was performed six months post-treatment. The generalized estimating equation test was used for statistical analysis. Acceptable levels of PDGF were detected in PLs (mean ± SD: 258.0 ± 52.3 pg/ml). The majority of horses (9/10) responded positively to PL treatment presenting lower lameness grades (p < 0.0005) compared to controls 10 days after the second injection, and returned to normal athletic activity. Radiographs revealed no changes in osteoarthritis lesions six months after treatment. One year post-injections, however, all horses relapsed to their initial degree of lameness. Intra-articularly injected autologous PL is an efficient method for temporarily managing osteoarthritis of the distal interphalangeal joint in athletic horses.

  2. Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation

    Directory of Open Access Journals (Sweden)

    Pasquale Marrazzo

    2016-01-01

    Full Text Available Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs. The perspective role of dental pulp stem cells (DPSCs in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1% was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H2O2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

  3. Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation.

    Science.gov (United States)

    Marrazzo, Pasquale; Paduano, Francesco; Palmieri, Francesca; Marrelli, Massimo; Tatullo, Marco

    2016-01-01

    Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H 2 O 2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

  4. Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

    Science.gov (United States)

    Dastan, Maryam; Najafzadeh, Nowruz; Abedelahi, Ali; Sarvi, Mohammadreza; Niapour, Ali

    2016-12-01

    Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and β-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100μM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and β-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (Pminoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (Pminoxidil-treated bulge cells. In contrast, the expression of β-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

    Science.gov (United States)

    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

  6. Engineering Enriched Microenvironments with Gradients of Platelet Lysate in Hydrogel Fibers.

    Science.gov (United States)

    Santo, Vítor E; Babo, Pedro; Amador, Miguel; Correia, Cláudia; Cunha, Bárbara; Coutinho, Daniela F; Neves, Nuno M; Mano, João F; Reis, Rui L; Gomes, Manuela E

    2016-06-13

    Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute toward morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photo-cross-linkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition, and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics, and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers toward the PL-enriched sections in comparison with the nonloaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions.

  7. The use of lumbar epidural injection of platelet lysate for treatment of radicular pain.

    Science.gov (United States)

    Centeno, Christopher; Markle, Jason; Dodson, Ehren; Stemper, Ian; Hyzy, Matthew; Williams, Christopher; Freeman, Michael

    2017-11-25

    Epidural steroid injections (ESI) are the most common pain management procedure performed in the US, however evidence of efficacy is limited. In addition, there is early evidence that the high dose of corticosteroids used can have systemic side effects. We describe the results of a case series evaluating the use of platelet lysate (PL) epidural injections for the treatment of lumbar radicular pain as an alternative to corticosteroids. Registry data was obtained for patients (N = 470) treated with PL epidural injections presenting with symptoms of lumbar radicular pain and MRI findings that were consistent with symptoms. Collected outcomes included numeric pain score (NPS), functional rating index (FRI), and a modified single assessment numeric evaluation (SANE) rating. Patients treated with PL epidurals reported significantly lower (p < .0001) NPS and FRI change scores at all time points compared to baseline. Post-treatment FRI change score means exceeded the minimal clinically important difference beyond 1 month. Average modified SANE ratings showed 49.7% improvement at 24 months post-treatment. Twenty-nine (6.3%) patients reported mild adverse events related to treatment. Patients treated with PL epidurals reported significant improvements in pain, exceeded the minimal clinically important difference (MCID) for FRI, and reported subjective improvement through 2-year follow-up. PL may be a promising substitute for corticosteroid.

  8. Heparin concentration is critical for cell culture with human platelet lysate.

    Science.gov (United States)

    Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

    2013-09-01

    Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Human platelet lysate supports the formation of robust human periodontal ligament cell sheets.

    Science.gov (United States)

    Tian, Bei-Min; Wu, Rui-Xin; Bi, Chun-Sheng; He, Xiao-Tao; Yin, Yuan; Chen, Fa-Ming

    2018-04-01

    The use of stem cell-derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet-induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix-related genes and proteins, such as collagen I, fibronectin and integrin β1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production. Copyright © 2017 John Wiley & Sons, Ltd.

  10. Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

    Science.gov (United States)

    Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

    2018-03-02

    Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

  11. Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

    Science.gov (United States)

    Dessels, Carla; Durandt, Chrisna; Pepper, Michael S

    2018-03-19

    Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

  12. Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future.

    Science.gov (United States)

    Astori, Giuseppe; Amati, Eliana; Bambi, Franco; Bernardi, Martina; Chieregato, Katia; Schäfer, Richard; Sella, Sabrina; Rodeghiero, Francesco

    2016-07-13

    The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing-thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2.PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.

  13. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    Directory of Open Access Journals (Sweden)

    Chengbo Tan

    2016-01-01

    Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

  14. Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

    Science.gov (United States)

    Allen, Ashley B; Butts, Emily B; Copland, Ian B; Stevens, Hazel Y; Guldberg, Robert E

    2017-10-01

    Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells.

    Science.gov (United States)

    Hassan, Ghmkin; Bahjat, Mohammad; Kasem, Issam; Soukkarieh, Chadi; Aljamali, Majd

    2018-01-01

    Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9 . Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

  16. Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Fioravanti, D; Bonanno, G; Totta, P; Zizzari, I G; Pierelli, L

    2016-08-25

    Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking

  17. Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use.

    Science.gov (United States)

    Wu, Rui-Xin; Yu, Yang; Yin, Yuan; Zhang, Xi-Yu; Gao, Li-Na; Chen, Fa-Ming

    2017-08-01

    Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future 'gold standard' supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue-specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either 'young' or 'old' donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of 'young' or 'old' PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS-expanded cells. PL- and FBS-expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL-expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells

    DEFF Research Database (Denmark)

    Glovinski, Peter Viktor; Herly, Mikkel; Mathiasen, Anders B

    2017-01-01

    , we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. METHODS: PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs...

  19. Bone Formation with Deproteinized Bovine Bone Mineral or Biphasic Calcium Phosphate in the Presence of Autologous Platelet Lysate: Comparative Investigation in Rabbit

    Directory of Open Access Journals (Sweden)

    Carole Chakar

    2014-01-01

    Full Text Available Bone substitutes alone or supplemented with platelet-derived concentrates are widely used to promote bone regeneration but their potency remains controversial. The aim of this study was, therefore, to compare the regenerative potential of preparations containing autologous platelet lysate (APL and particles of either deproteinized bovine bone mineral (DBBM or biphasic calcium phosphate (BCP, two bone substitutes with different resorption patterns. Rabbit APL was prepared by freeze-thawing a platelet suspension. Critical-size defects in rabbit femoral condyle were filled with DBBM or DBBM+APL and BCP or BCP+APL. Rabbits were sacrificed after six weeks and newly formed bone and residual implanted material were evaluated using nondemineralized histology and histomorphometry. New bone was observed around particles of all fillers tested. In the defects filled with BCP, the newly formed bone area was greater (70%; P<0.001 while the residual material area was lower (60%; P<0.001 than that observed in those filled with DBBM. New bone and residual material area of defects filled with either APL+DBBM or APL+BCP were similar to those observed in those filled with the material alone. In summary, osteoconductivity and resorption of BCP were greater than those of DBBM, while APL associated with either DBBM or BCP did not have an additional benefit.

  20. Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

    Science.gov (United States)

    Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

    2013-12-01

    Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  1. Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

    Science.gov (United States)

    Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

    2013-01-01

    Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

  2. Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress.

    Science.gov (United States)

    Homayouni Moghadam, Farshad; Tayebi, Tahereh; Moradi, Alireza; Nadri, Hamid; Barzegar, Kazem; Eslami, Gilda

    2014-01-01

    By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm(2)) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm(2)) for a long time (24 hours) or high tension (10 dyn/cm(2)) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel.

  3. Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue

    DEFF Research Database (Denmark)

    Juhl, Morten; Tratwal, Josefine; Follin, Bjarke

    2016-01-01

    be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. METHODS: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three...... culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. RESULTS: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS...

  4. Innate immune responses of equine monocytes cultured in equine platelet lysate.

    Science.gov (United States)

    Naskou, Maria C; Norton, Natalie A; Copland, Ian B; Galipeau, Jacques; Peroni, John F

    2018-01-01

    Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1β (IL-1β) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1β. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1β production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1β compared to the control groups. This is the first study to demonstrate that ePL suppresses

  5. The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

    Science.gov (United States)

    Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

    2016-04-01

    Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

  6. Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

    Science.gov (United States)

    Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

    2017-09-09

    Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture

    International Nuclear Information System (INIS)

    Yoshito, Daniele

    2011-01-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  8. Sponge-Like Dressings Based on the Association of Chitosan and Sericin for the Treatment of Chronic Skin Ulcers. II. Loading of the Hemoderivative Platelet Lysate.

    Science.gov (United States)

    Mori, Michela; Rossi, Silvia; Ferrari, Franca; Bonferoni, Maria C; Sandri, Giuseppina; Riva, Federica; Tenci, Marika; Del Fante, Claudia; Nicoletti, Giovanni; Caramella, Carla

    2016-03-01

    Platelet lysate (PL) was loaded into dressings based on chitosan glutamate (CSG) low and high molecular weight, sericin (Ser), and glycine (Gly). A synergic effect of Ser and PL on fibroblast proliferation was proved in vitro. Two different PL loading approaches were considered: the first provided to prepare dressings by freeze-drying a mixture of PL and CSG/Gly/Ser solution, the second approach consisted in the extemporarily loading of PL in the CSG/Gly/Ser freeze-dried dressings. As for the first approach, PL loading did not produce any variation in dressing mechanical properties. Such dressings absorbed a high amount (about 8-fold of dry weight) of phosphate-buffered saline (fluid mimicking wound exudate), forming a gel with pseudoplastic and elastic properties. Platelet-derived growth factor AB assay indicated that neither freeze-drying nor the excipients alter PL growth factor content. As for the second approach, mechanical and rheological properties of the gel formed upon PL absorption enabled to choose a PL loading of about 90 μL/cm(2). Upon contact with fibroblasts, all PL loaded formulations increased the number not only of viable cells but also of those in the proliferative phase. Histological studies effected on human skin strips pointed out the positive effect of PL loaded dressings on dermal matrix reconstruction. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  9. Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

    Science.gov (United States)

    Fernandez-Rebollo, Eduardo; Mentrup, Birgit; Ebert, Regina; Franzen, Julia; Abagnale, Giulio; Sieben, Torsten; Ostrowska, Alina; Hoffmann, Per; Roux, Pierre-François; Rath, Björn; Goodhardt, Michele; Lemaitre, Jean-Marc; Bischof, Oliver; Jakob, Franz; Wagner, Wolfgang

    2017-07-11

    Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

  10. Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

    NARCIS (Netherlands)

    Fijnheer, R.; Pietersz, R. N.; de Korte, D.; Gouwerok, C. W.; Dekker, W. J.; Reesink, H. W.; Roos, D.

    1990-01-01

    The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from

  11. Evaluation of a platelet lysate bilayered system for periodontal regeneration in a rat intrabony three-wall periodontal defect.

    Science.gov (United States)

    Babo, Pedro S; Cai, Xinjie; Plachokova, Adelina S; Reis, Rui L; Jansen, John; Gomes, Manuela E; Walboomers, X Frank

    2018-02-01

    With currently available therapies, full regeneration of lost periodontal tissues after periodontitis cannot be achieved. In this study, a combined compartmentalized system was tested, composed of (a) a platelet lysate (PL)-based construct, which was placed along the root aiming to regenerate the root cementum and periodontal ligament, and (b) a calcium phosphate cement composite incorporated with hyaluronic acid microspheres loaded with PL, aiming to promote the regeneration of alveolar bone. This bilayered system was assessed in a 3-wall periodontal defect in Wistar rats. The periodontal healing and the inflammatory response of the materials were scored for a period up to 6 weeks after implantation. Furthermore, histomorphometrical measurements were performed to assess the epithelial downgrowth, the formation of alveolar bone, and the formation of new connective tissue attachment. Our data showed that the stabilization of platelet-origin proteins on the root surface increased the overall periodontal healing score and restricted the formation of long epithelial junctions. Nevertheless, the faster degradation of the cement component with incorporated hyaluronic acid microspheres compromised the stability of the system, which hampered the periodontal regeneration. Overall, in this work, we proved the positive therapeutic effect of the immobilization of a PL-based construct over the root surface in a combined compartmentalized system to assist predictable healing of functional periodontium. Therefore, after optimization of the hard tissue analogue, the system should be further elaborated in (pre)clinical validation studies. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Differentiation of Rat bone marrow Mesenchymal stem cells into Adipocytes and Cardiomyocytes after treatment with platelet lysate.

    Science.gov (United States)

    Homayouni Moghadam, Farshad; Tayebi, Tahereh; Barzegar, Kazem

    2016-01-01

    Mesenchymal stem cells (MSCs) are multipotential cells and their therapeutic potency is under intense investigation. Studying the effect of different induction factors on MSCs could increase our knowledge about the differentiation potency of these cells. One of the most important sources of these factors in mammalian body is platelet. Platelet lysate (PL) contains many growth factors and therefore, it can be used as a differentiation inducer. In the present study, the effect of PL on differentiation of rat bone marrow MSCs into cardiomyocytes was studied. To study the differentiation-inducing effect of PL, MSCs were treated with 2.5, 5 and 10% PL. Early results of this study showed that PL in high concentrations (10%) induces adipogenic differentiation of MSCs. Therefore, to evaluate differentiation to cardiomyocytes, MSCs were cultured in media containing lower levels of PL (2.5% and 5%) and then cardiomyogenic differentiation was induced by treatment with 5-azacytidine. Differentiation of MSCs was evaluated using direct observation of beating cells, immunostaining and real-time PCR techniques. The results of qPCR showed that treatment with PL alone increased the expression of cardiac alpha actinin (CAA) being predictable by earlier observation of beating cells in PL-treated groups. The results of staining assays against cardiac alpha actinin also showed that there were stained cells in PL-treated groups. The results of the present study showed that PL is a powerful induction factor for differentiation of MSCs into different cell lines such as cardiomyocytes and adipocytes.

  13. Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

    Science.gov (United States)

    Nguyen, Van Thi; Cancedda, Ranieri; Descalzi, Fiorella

    2018-03-01

    The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage. Copyright © 2017 John Wiley & Sons, Ltd.

  14. A novel dressing for the combined delivery of platelet lysate and vancomycin hydrochloride to chronic skin ulcers: Hyaluronic acid particles in alginate matrices.

    Science.gov (United States)

    Rossi, S; Mori, M; Vigani, B; Bonferoni, M C; Sandri, G; Riva, F; Caramella, C; Ferrari, F

    2018-06-15

    The aim of the present work was to develop a medication allowing for the combined delivery of platelet lysate (PL) and an anti-infective model drug, vancomycin hydrochloride (VCM), to chronic skin ulcers. A simple method was set up for the preparation of hyaluronic acid (HA) core-shell particles, loaded with PL and coated with calcium alginate, embedded in a VCM containing alginate matrix. Two different CaCl 2 concentrations were investigated to allow for HA/PL core-shell particle formation. The resulting dressings were characterized for mechanical and hydration properties and tested in vitro (on fibroblasts) and ex-vivo (on skin biopsies) for biological activity. They were found of sufficient mechanical strength to withstand packaging and handling stress and able to absorb a high amount of wound exudate and to form a protective gel on the lesion area. The CaCl 2 concentration used for shell formation did not affect VCM release from the alginate matrix, but strongly modified the release of PGFAB (chosen as representative of growth factors present in PL) from HA particles. In vitro and ex vivo tests provided sufficient proof of concept of the ability of dressings to improve skin ulcers healing. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Particulate systems based on pectin/chitosan association for the delivery of manuka honey components and platelet lysate in chronic skin ulcers.

    Science.gov (United States)

    Tenci, Marika; Rossi, Silvia; Bonferoni, Maria Cristina; Sandri, Giuseppina; Boselli, Cinzia; Di Lorenzo, Arianna; Daglia, Maria; Icaro Cornaglia, Antonia; Gioglio, Luciana; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

    2016-07-25

    The aim of the present work was the development of a powder formulation for the delivery of manuka honey (MH) bioactive components and platelet lysate (PL) in chronic skin ulcers. In particular pectin (PEC)/chitosan (CS) particles were prepared by ionotropic gelation in the presence of calcium chloride and subsequently characterized for particle size, hydration properties and mechanical resistance. Different experimental conditions (calcium chloride and CS concentrations; rest time in the cationic solution) were considered in order to obtain particles characterized by optimal size, hydration properties and mechanical resistance. Two different fractions of MH were examined: one (Fr1), rich in methylglyoxal and the other (Fr2), rich in polyphenols. Particles were loaded with Fr1, fraction able to enhance in vitro proliferation of human fibroblasts, and with PL. The presence of CS in Fr1-loaded particles produced an improvement in cell proliferation. Moreover, PL loading into particles did not affect the biological activity of the hemoderivative. In vivo efficacy of PL- and Fr1-loaded particles was evaluated on a rat wound model. Both treatments markedly increased wound healing to the same extent. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Development of an Injectable Calcium Phosphate/Hyaluronic Acid Microparticles System for Platelet Lysate Sustained Delivery Aiming Bone Regeneration.

    Science.gov (United States)

    Babo, Pedro S; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L

    2016-11-01

    Despite the biocompatibility and osteoinductive properties of calcium phosphate (CaP) cements their low biodegradability hampers full bone regeneration. Herein the incorporation of CaP cement with hyaluronic acid (HAc) microparticles loaded with platelet lysate (PL) to improve the degradability and biological performance of the cements is proposed. Cement formulations incorporating increasing weight ratios of either empty HAc microparticles or microparticles loaded with PL (10 and 20 wt%) are developed as well as cements directly incorporating PL. The direct incorporation of PL improves the mechanical properties of the plain cement, reaching values similar to native bone. Morphological analysis shows homogeneous particle distribution and high interconnectivity between the HAc microparticles. The cements incorporating PL (with or without the HAc microparticles) present a sustained release of PL proteins for up to 8 d. The sustained release of PL modulates the expression of osteogenic markers in seeded human adipose tissue derived stem cells, thus suggesting the stimulatory role of this hybrid system toward osteogenic commitment and bone regeneration applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

    Science.gov (United States)

    Le Pape, Fiona; Cosnuau-Kemmat, Lucie; Richard, Gaëlle; Dubrana, Frédéric; Férec, Claude; Zal, Franck; Leize, Elisabeth; Delépine, Pascal

    2017-04-01

    Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  18. The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

    Science.gov (United States)

    Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

    2013-12-01

    Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Long-term safety and efficacy of autologous platelet lysate drops for treatment of ocular GvHD.

    Science.gov (United States)

    Pezzotta, S; Del Fante, C; Scudeller, L; Rossi, G C; Perotti, C; Bianchi, P E; Antoniazzi, E

    2017-01-01

    Current ocular GvHD (oGvHD) treatments are suboptimal. We investigated the safety and efficacy of long-term continuous treatment with autologous platelet lysate (PL) drops in patients with oGvHD Dry Eye Syndrome (DES) score 2-3 refractory to topical conventional therapy. Ophthalmic evaluation was performed at 6 month intervals. Symptoms were assessed using the Glaucoma Symptom Scale (GSS). Patients were defined 'responders' when showing a reduction at least one grade on National Institutes of Health Eye Score from baseline at the 6 month visit. Thirty-one patients were included, and 16 (51%) completed 36 months of follow-up (range 6.5-72.7). At 6 months all patients were classified as responders: median GSS symptom score decreased from 70 to 41 (33 at 36 months), median GSS function score reduced from 68 to 46 (33 at 36 months) (all P<0.001). Median Tear Break Up Time improved from 3 to 6 s after 6 months and was maintained over time. All signs improved at 6 and 36 months (clinical and statistical significance). No severe adverse events occurred. Long-term treatment with PL drops is secure and effective for oGvHD and can be an efficient therapy option from initial stages of oGvHD to prevent permanent ocular impairment and improving quality of life.

  20. Evaluation of Peripheral Blood and Cord Blood Platelet Lysates in Isolation and Expansion of Multipotent Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Ioanna Christou

    2018-02-01

    Full Text Available Background: Multipotent Mesenchymal Stromal Cells (MSCs are used in tissue engineering and regenerative medicine. The in vitro isolation and expansion of MSCs involve the use of foetal bovine serum (FBS. However, many concerns have been raised regarding the safety of this product. In this study, alternative additives derived either from peripheral or cord blood were tested as an FBS replacement. Methods: Platelet lysates (PL from peripheral and cord blood were used for the expansion of MSCs. The levels of growth factors in peripheral blood (PB and cord blood (CB PLs were determined using the Multiple Reaction Monitoring (MRM. Finally, the cell doubling time (CDT, tri-lineage differentiation and phenotypic characterization of the MSCs expanded with FBS and PLs were determined. Results: MSCs treated with culture media containing FBS and PB-PL, were successfully isolated and expanded, whereas MSCs treated with CB-PL could not be maintained in culture. Furthermore, the MRM analysis yielded differences in growth factor levels between PB-PL and CB-PL. In addition, the MSCs were successfully expanded with FBS and PB-PL and exhibited tri-lineage differentiation and stable phenotypic characteristics. Conclusion: PB-PL could be used as an alternative additive for the production of MSCs culture medium applied to xenogeneic-free expansion and maintenance of MSCs in large scale clinical studies.

  1. Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

    Science.gov (United States)

    Kim, Hyojin; Prasain, Nutan; Vemula, Sasidhar; Ferkowicz, Michael J; Yoshimoto, Momoko; Voytik-Harbin, Sherry L; Yoder, Mervin C

    2015-09-01

    Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Mesenchymal Stromal Cells Implantation in Combination with Platelet Lysate Product Is Safe for Reconstruction of Human Long Bone Nonunion.

    Science.gov (United States)

    Labibzadeh, Narges; Emadedin, Mohsen; Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

    2016-01-01

    Nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion. In this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period. From a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control Xray evidence, bony union had occurred. Results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179).

  3. Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia.

    Science.gov (United States)

    Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd; Juhl, Morten; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana

    2017-01-01

    Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

    Science.gov (United States)

    Švajger, Urban

    2017-04-01

    Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  5. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy.

    Science.gov (United States)

    Sykes, J G; Kuiper, J H; Richardson, J B; Roberts, S; Wright, K T; Kuiper, N J

    2018-05-01

    High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI). The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS) and Stemulate™ (a commercial source of human platelet lysate), on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM) vs. Stemulate™, 13.10 ± 2.57 d (SEM)]. Sulphated glycosaminoglycans (sGAG), total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype) and, hence, for cell therapies (including ACI).

  6. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy

    Directory of Open Access Journals (Sweden)

    JG Sykes

    2018-05-01

    Full Text Available High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI. The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS and Stemulate™ (a commercial source of human platelet lysate, on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM vs. Stemulate™, 13.10 ± 2.57 d (SEM]. Sulphated glycosaminoglycans (sGAG, total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype and, hence, for cell therapies (including ACI.

  7. Human platelet lysate in mesenchymal stromal cell expansion according to a GMP grade protocol: a cell factory experience.

    Science.gov (United States)

    Becherucci, Valentina; Piccini, Luisa; Casamassima, Serena; Bisin, Silvia; Gori, Valentina; Gentile, Francesca; Ceccantini, Riccardo; De Rienzo, Elena; Bindi, Barbara; Pavan, Paola; Cunial, Vanessa; Allegro, Elisa; Ermini, Stefano; Brugnolo, Francesca; Astori, Giuseppe; Bambi, Franco

    2018-05-02

    The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production.

  8. Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

    Science.gov (United States)

    Griffiths, Sarah; Baraniak, Priya R; Copland, Ian B; Nerem, Robert M; McDevitt, Todd C

    2013-12-01

    Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown. MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to "rescue" the proliferative capacity of MSCs. hPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS. hPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

    Science.gov (United States)

    Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

    2018-05-01

    Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

  10. Comparison of point-of-care methods for preparation of platelet concentrate (platelet-rich plasma).

    Science.gov (United States)

    Weibrich, Gernot; Kleis, Wilfried K G; Streckbein, Philipp; Moergel, Maximilian; Hitzler, Walter E; Hafner, Gerd

    2012-01-01

    This study analyzed the concentrations of platelets and growth factors in platelet-rich plasma (PRP), which are likely to depend on the method used for its production. The cellular composition and growth factor content of platelet concentrates (platelet-rich plasma) produced by six different procedures were quantitatively analyzed and compared. Platelet and leukocyte counts were determined on an automatic cell counter, and analysis of growth factors was performed using enzyme-linked immunosorbent assay. The principal differences between the analyzed PRP production methods (blood bank method of intermittent flow centrifuge system/platelet apheresis and by the five point-of-care methods) and the resulting platelet concentrates were evaluated with regard to resulting platelet, leukocyte, and growth factor levels. The platelet counts in both whole blood and PRP were generally higher in women than in men; no differences were observed with regard to age. Statistical analysis of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor β1 (TGF-β1) showed no differences with regard to age or gender. Platelet counts and TGF-β1 concentration correlated closely, as did platelet counts and PDGF-AB levels. There were only rare correlations between leukocyte counts and PDGF-AB levels, but comparison of leukocyte counts and PDGF-AB levels demonstrated certain parallel tendencies. TGF-β1 levels derive in substantial part from platelets and emphasize the role of leukocytes, in addition to that of platelets, as a source of growth factors in PRP. All methods of producing PRP showed high variability in platelet counts and growth factor levels. The highest growth factor levels were found in the PRP prepared using the Platelet Concentrate Collection System manufactured by Biomet 3i.

  11. Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate.

    Science.gov (United States)

    Heathman, Thomas R J; Stolzing, Alexandra; Fabian, Claire; Rafiq, Qasim A; Coopman, Karen; Nienow, Alvin W; Kara, Bo; Hewitt, Christopher J

    2016-04-01

    The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 10(5) and 1.02 ± 0.005 × 10(5) cells/mL compared with 0.79 ± 0.05 × 10(5) and 0.36 ± 0.04 × 10(5) cells/mL in FBS-containing medium. This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. Platelet collection efficiencies of three different platelet-rich plasma preparation systems.

    Science.gov (United States)

    Aydin, Fatma; Pancar Yuksel, Esra; Albayrak, Davut

    2015-06-01

    Different systems have been used for the preparation of platelet-rich plasma (PRP), but platelet collection efficiencies of these systems are not clear. To evaluate the platelet collection efficiencies of three different PRP preparation systems. Blood samples were obtained from the same 16 volunteers for each system. The samples were centrifuged and PRP was prepared by three systems. The ratio of the total number of platelets in PRP to the total number of platelets of the venous blood sample of the patient expressed in percentage was named as platelet collection efficiency and calculated for each system. Mean platelet collection efficiencies were 66.6 (min: 56.9, max: 76.9), 58.3 (min: 27.3, max: 102.8), 50.8 (min: 27.2, max: 73) for top and bottom bag system, system using citrated tube, and the system using tube with Ficoll and cell extraction kit, respectively. Statistically significant difference was found only between the platelet collection efficiencies of systems using the tube with ficoll and cell extraction kit and the top and bottom bag system (p = 0.002). All three systems could be used for PRP preparation, but top and bottom bag system offers a slight advantage over the system using Ficoll and cell extraction kit regarding the platelet collection efficiency.

  13. Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Bernardi, Martina; Albiero, Elena; Alghisi, Alberta; Chieregato, Katia; Lievore, Chiara; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2013-08-01

    A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  14. The Role of a Platelet Lysate-Based Compartmentalized System as a Carrier of Cells and Platelet-Origin Cytokines for Periodontal Tissue Regeneration.

    Science.gov (United States)

    Babo, Pedro S; Cai, Xinjie; Plachokova, Adelina S; Reis, Rui L; Jansen, John A; Gomes, Manuela E; Walboomers, X Frank

    2016-10-01

    Currently available clinical therapies are not capable to regenerate tissues that are lost by periodontitis. Tissue engineering can be applied as a strategy to regenerate reliably the tissues and function of damaged periodontium. A prerequisite for this regeneration is the colonization of the defect with the adequate cell populations. In this study, we proposed a bilayered system composed of (1) a platelet lysate (PL)-based construct produced by crosslinking of PL proteins with genipin (gPL) for the delivery of rat periodontal ligament cells (rat-PDLCs); combined with (2) an injectable composite consisting of calcium phosphate cement incorporated with PL-loaded poly(d, l-lactic-co-glycolic acid) microspheres. This system was expected to promote periodontal regeneration by the delivery of adequate progenitor cells and providing a stable system enriched with adequate cytokines and growth factors for the orchestration of tissue regrowth in periodontal defects. The bilayered system was tested in a three-wall intrabony defect in rats and the healing of periodontal tissue was assessed 6 weeks after surgery. Results showed that the bilayered system was able to promote the regrowth of functional periodontal tissues, both with (cells + gPL) and without the loading of PDLCs (gPL). Significant connective tissue attachment (45.0 ± 15.0% and 64.0 ± 15.0% for gPL and cells + gPL group, respectively) and new bone area (33.8 ± 21% and 21.3 ± 3% for gPL and cells + gPL group, respectively) were observed. Nevertheless, rat PDLCs delivered with gPL construct in the defect area were hardly visible 6 weeks after surgery and did not contribute for the regeneration of new periodontal tissue. Overall, our findings show that the bilayered system promotes the stabilization of PL proteins on the root surface and has a positive effect in the repair of periodontal tissues both in quality and in quantity.

  15. Comparison of osteo/odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate.

    Science.gov (United States)

    Abuarqoub, Duaa; Awidi, Abdalla; Abuharfeil, Nizar

    2015-10-01

    Human dental pulp cells (DPSCs) and stem cells from apical papilla have been used for the repair of damaged tooth tissues. Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) for large scale expansion of dental stem cells. However, biological effects and optimal concentrations of PL for proliferation and differentiation of human dental stem cells remain to be elucidated. DPSCs and SCAP cells were isolated from impacted third molars of young healthy donors, at the stage of root development and identified by markers using flow cytometry. For comparison the cells were cultured in media containing PL (1%, 5% and 10%) and FBS, with subsequent induction for osteogenic/odontogenic differentiation. The cultures were analyzed for; morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers using ELISA and real time -polymerase chain reaction (qPCR). The proliferation rates of DPSCs and SCAP significantly increased when cells were treated with 5% PL (7X doubling time) as compared to FBS. 5% PL also enhanced mineralized differentiation of DPSCs and SCAP, as indicated by the measurement of alkaline phosphatase activity, osteocalcin and osteopontin, calcium deposition and q-PCR. Our findings suggest that using 5% platelet lysate, proliferation and osteo/odontogenesis of DPSCs and SCAP for a short period of time (15 days), was significantly improved. This may imply its use as an optimum concentration for expansion of dental stem cells in bone regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Human Platelet Lysate as a Xeno Free Alternative of Fetal Bovine Serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells.

    Science.gov (United States)

    Mohammadi, Saeed; Nikbakht, Mohsen; Malek Mohammadi, Ashraf; Zahed Panah, Mahdi; Ostadali, Mohammad Reza; Nasiri, Hajar; Ghavamzadeh, Ardeshir

    2016-07-01

    Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives. Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR. We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement. We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability.

  17. Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

    Science.gov (United States)

    Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

    2018-05-02

    Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our

  18. Development of chitosan oleate ionic micelles loaded with silver sulfadiazine to be associated with platelet lysate for application in wound healing.

    Science.gov (United States)

    Dellera, Eleonora; Bonferoni, Maria Cristina; Sandri, Giuseppina; Rossi, Silvia; Ferrari, Franca; Del Fante, Claudia; Perotti, Cesare; Grisoli, Pietro; Caramella, Carla

    2014-11-01

    In the treatment of chronic wounds, topical application of anti-infective drugs such as silver sulfadiazine (AgSD) is of primary importance to avoid infections and accelerate wound repair. AgSD is used in burns and chronic wounds for its wide antibacterial spectrum, but presents limitations due to poor solubility and cytotoxicity. In the present work polymeric micelles obtained by self-assembling of chitosan ionically modified by interaction with oleic acid were developed as carriers for AgSD to overcome the drawbacks of the drug. The AgSD loaded micelles were intended to be associated in wound healing with platelet lysate (PL), a hemoderivative rich in growth factors. Unloaded micelles demonstrated good compatibility with both fibroblasts and PL. The relevance of chitosan concentration and of the ratio between chitosan and oleic acid to the drug loading and the particle size of nanoparticles was studied. A marked increase (up to 100 times with respect to saturated solution) of AgSD concentration in micelle dispersion was obtained. Moreover, the encapsulation reduced the cytotoxic effect of the drug towards fibroblasts and the drug incompatibility with PDGF-AB (platelet derived growth factor), chosen as representative of platelet growth factors. Copyright © 2014. Published by Elsevier B.V.

  19. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

    Science.gov (United States)

    Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

    2009-01-01

    Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA

  20. Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

    Science.gov (United States)

    de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

    2017-05-01

    Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  1. A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

    OpenAIRE

    Mustafa Tunalı; Hakan Özdemir; Zafer Küçükodacı; Serhan Akman; Emre Yaprak; Hülya Toker; Erhan Fıratlı

    2014-01-01

    We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn us...

  2. Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

    Science.gov (United States)

    Russell, K A; Koch, T G

    2016-03-01

    Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function. © 2015 EVJ Ltd.

  3. Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Sabrina Viau

    Full Text Available We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL that constitutes an advantageous substitute for fetal bovine serum (FBS for human mesenchymal stem cell (hMSC expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological and ethical issues. Because of the progressive use of pathogen-reduced (PR labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content and efficacy (as a medium supplement for hMSC expansion. This technology is based on short-wave ultraviolet light (UV-C that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs.We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1 but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01. We performed large-scale culture of hMSCs from bone marrow (BM during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel

  4. Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Viau, Sabrina; Chabrand, Lucie; Eap, Sandy; Lorant, Judith; Rouger, Karl; Goudaliez, Francis; Sumian, Chryslain; Delorme, Bruno

    2017-01-01

    We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs). We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the

  5. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Camilla Siciliano

    2015-01-01

    Full Text Available Human adipose tissue-derived mesenchymal stem cells (ADMSCs are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL, a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

  6. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

    2015-01-01

    Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. PMID:26495284

  7. Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

    2018-03-22

    Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

  8. Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion.

    Science.gov (United States)

    Kao, Yu-Chun; Bailey, Andy; Samminger, Bernhard; Tanimoto, Junji; Burnouf, Thierry

    2016-07-01

    Pooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. Frozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. QSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. QSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells

    Science.gov (United States)

    Russell, Keith A.; Gibson, Thomas W. G.; Chong, Andrew; Co, Carmon; Koch, Thomas G.

    2015-01-01

    Background Mesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC. Hypothesis and Objectives We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation. Results 1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis. Conclusions/Significance Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects. PMID:26353112

  10. Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

    Directory of Open Access Journals (Sweden)

    Keith A Russell

    Full Text Available Mesenchymal stromal cells (MSC are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL has proven to be a suitable alternative to FBS for expansion of human MSC.We hypothesized that canine adipose tissue (AT and bone marrow (BM MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1 isolation, 2 proliferation, 3 spontaneous differentiation, and 4 directed differentiation.1 Medium with 10% PL was unable to isolate MSC. 2 MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT or 30% (BM enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3 Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4 MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis.Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.

  11. Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

    Science.gov (United States)

    Tasev, Dimitar; van Wijhe, Michiel H; Weijers, Ester M; van Hinsbergh, Victor W M; Koolwijk, Pieter

    2015-01-01

    Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. The presented isolation method and subsequent cell expansion in platelet lysate

  12. Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

    Science.gov (United States)

    Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

    2018-02-01

    The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.

  13. Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

    Directory of Open Access Journals (Sweden)

    Dimitar Tasev

    Full Text Available Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.The findings presented in this study indicate that human platelet lysate (PL as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS. Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator or uPAR (uPA receptor by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.The presented isolation method and subsequent cell expansion in platelet

  14. Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC and apheresis-PC methods

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    Singh Ravindra

    2009-01-01

    Full Text Available Background: Platelet rich plasma-platelet concentrate (PRP-PC, buffy coat poor-platelet concentrate (BC-PC, and apheresis-PC were prepared and their quality parameters were assessed. Study Design: In this study, the following platelet products were prepared: from random donor platelets (i platelet rich plasma - platelet concentrate (PRP-PC, and (ii buffy coat poor- platelet concentrate (BC-PC and (iii single donor platelets (apheresis-PC by different methods. Their quality was assessed using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH. Results: A total of 146 platelet concentrates (64 of PRP-PC, 62 of BC-PC and 20 of apheresis-PC were enrolled in this study. The mean volume of PRP-PC, BC-PC and apheresis-PC was 62.30±22.68 ml, 68.81±22.95 ml and 214.05±9.91 ml and ranged from 22-135 ml, 32-133 ml and 200-251 ml respectively. The mean platelet count of PRP-PC, BC-PC and apheresis-PC was 7.6±2.97 x 1010/unit, 7.3±2.98 x 1010/unit and 4.13±1.32 x 1011/unit and ranged from 3.2-16.2 x 1010/unit, 0.6-16.4 x 1010/unit and 1.22-8.9 x 1011/unit respectively. The mean WBC count in PRP-PC (n = 10, BC-PC (n = 10 and apheresis-PC (n = 6 units was 4.05±0.48 x 107/unit, 2.08±0.39 x 107/unit and 4.8±0.8 x 106/unit and ranged from 3.4 -4.77 x 107/unit, 1.6-2.7 x 107/unit and 3.2 - 5.2 x 106/unit respectively. A total of 26 units were analyzed for pH changes. Out of these units, 10 each were PRP-PC and BC-PC and 6 units were apheresis-PC. Their mean pH was 6.7±0.26 (mean±SD and ranged from 6.5 - 7.0 and no difference was observed among all three types of platelet concentrate. Conclusion: PRP-PC and BC-PC units were comparable in terms of swirling, platelet count per unit and pH. As expected, we found WBC contamination to be less in BC-PC than PRP-PC units. Variation in volume was more in BC-PC than PRP-PC units and this suggests that further standardization is required for preparation of BC

  15. Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

    Science.gov (United States)

    Davis, Vicki L; Abukabda, Alaeddin B; Radio, Nicholas M; Witt-Enderby, Paula A; Clafshenkel, William P; Cairone, J Vito; Rutkowski, James L

    2014-08-01

    Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-β1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

  16. Pooled human platelet lysate versus fetal bovine serum—investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use

    DEFF Research Database (Denmark)

    Trojahn Kølle, Stig-Frederik; Oliveri, Roberto S; Glovinski, Peter V

    2013-01-01

    Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) ......) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS))....

  17. A novel platelet concentrate: titanium-prepared platelet-rich fibrin.

    Science.gov (United States)

    Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Yaprak, Emre; Toker, Hülya; Fıratlı, Erhan

    2014-01-01

    We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF) was processed with a scanning electron microscope (SEM). The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  18. A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

    Directory of Open Access Journals (Sweden)

    Mustafa Tunalı

    2014-01-01

    Full Text Available We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF. The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun’s leukocyte- and platelet-rich fibrin (L-PRF method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF was processed with a scanning electron microscope (SEM. The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

  19. Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate.

    Science.gov (United States)

    Wagner, Eric R; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M; Chase, Steven; Westendorf, Jennifer J; Dietz, Allan B; van Wijnen, Andre J; Yaszemski, Michael J; Kakar, Sanjeev

    2015-11-01

    Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer "neoligament" seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell-cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p engineering and ligament regeneration.

  20. Comparison of allogeneic platelet lysate and fetal bovine serum for in vitro expansion of equine bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Seo, Jong-pil; Tsuzuki, Nao; Haneda, Shingo; Yamada, Kazutaka; Furuoka, Hidefumi; Tabata, Yasuhiko; Sasaki, Naoki

    2013-10-01

    Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy and tissue engineering approaches. Fetal bovine serum (FBS) is commonly used for in vitro MSC expansion; however, the use of FBS may be associated with ethical, scientific, and safety issues. This study aimed to compare the ability of allogeneic platelet lysate (PL) and FBS to cause equine bone marrow-derived MSC expansion. MSCs were isolated from bone marrow aspirate in media supplemented with either PL or FBS, and cell proliferation properties and characteristics were examined. There were no significant differences in MSC yield, colony-forming unit-fibroblast (CFU-F) assay, and population doubling time between PL and FBS cultures. In addition, both PL-MSCs and FBS-MSCs showed similar results in term of ALP staining, osteogenic differentiation, and RT-PCR, although there were subtle differences in morphology, growth pattern, and adhesive properties. These results suggest that PL is a suitable alternative to FBS for use in equine MSC expansion, without the problems related to FBS use. Published by Elsevier India Pvt Ltd.

  1. Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum

    Science.gov (United States)

    2012-01-01

    Introduction Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM) but potentially from umbilical cord blood (UCB). Although they are becoming a good tool for regenerative medicine, they usually need to be expanded in fetal bovine serum (FBS)-supplemented media. Human platelet lysate (HPL) has recently been proposed as substitute for safety reasons, but it is not yet clear how this supplement influences the properties of expanded MSCs. Methods In the present study, we compared the effect of various media combining autologous HPL with or without FBS on phenotypic, proliferative and functional (differentiation, cytokine secretion profile) characteristics of human BM-derived MSCs. Results Despite less expression of adipogenic and osteogenic markers, MSCs cultured in HPL-supplemented media fully differentiated along osteoblastic, adipogenic, chondrogenic and vascular smooth muscle lineages. The analyses of particular specific proteins expressed during osteogenic differentiation (calcium-sensing receptor (CaSR) and parathormone receptor (PTHR)) showed their decrease at D0 before any induction for MSC cultured with HPL mostly at high percentage (10%HPL). The cytokine dosage showed a clear increase of proliferation capacity and interleukin (IL)-6 and IL-8 secretion. Conclusions This study shows that MSCs can be expanded in media supplemented with HPL that can totally replace FBS. HPL-supplemented media not only preserves their phenotype as well as their differentiation capacity, but also shortens culture time by increasing their growth rate. PMID:22333342

  2. Autologous blood preparations rich in platelets, fibrin and growth factors.

    Science.gov (United States)

    Fioravanti, C; Frustaci, I; Armellin, E; Condò, R; Arcuri, C; Cerroni, L

    2015-01-01

    Bone regeneration is often needed prior to dental implant treatment due to the lack of adequate quantity and quality after infectious diseases. The greatest regenerative power was obtained with autologous tissue, primarily the bone alive, taken from the same site or adjacent sites, up to the use centrifugation of blood with the selection of the parts with the greatest potential regenerative. In fact, various techniques and technologies were chronologically successive to cope with an ever better preparation of these concentrates of blood. Our aim is to review these advances and discuss the ways in which platelet concentrates may provide such unexpected beneficial therapeutic effects. The research has been carried out in the MEDLINE and Cochrane Central Register of Controlled Trials database by choosing keywords as "platelet rich plasma", "platelet rich fibrin", "platelet growth factors", and "bone regeneration" and "dentistry". Autologous platelet rich plasma is a safe and low cost procedure to deliver growth factors for bone and soft tissue healing. The great heterogeneity of clinical outcomes can be explained by the different PRP products with qualitative and quantitative difference among substance.

  3. Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures.

    Science.gov (United States)

    Noulsri, Egarit; Udomwinijsilp, Prapaporn; Lerdwana, Surada; Chongkolwatana, Viroje; Permpikul, Parichart

    2017-04-01

    There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. A contribution to the study of pyrogenic substances in radiopharmaceutical preparations. Comparison between methods using Rabbit and those using Limulus amebocytes lysate

    International Nuclear Information System (INIS)

    Bruneau, Jacky.

    1982-10-01

    We have studied two methods for pyrogenic substances detection. We used: the hyperthermic action of these substances after injection in Rabbit, and the gelation reaction of a Limulus amebocytes lysate. To apply these two methods of pyrogenic substances detection to the radiopharmaceutical preparations, we have conceived and designed a material allowing their handling in compliance with the radioactive safety norms. We have compared the sensitivity, reliability and reproducibility of these methods, one based on gelation of Limulus amebocytes lysate in presence of endotoxins, the other on the hyperthermic action of these same endotoxins in the rabbit when injected intravenously or through the suboccipital route. The discussion of the results obtained, shows that the method using the Limulus amebocytes lysate is more sensitive, less expansive and less dangerous. This method particulary well adapted to the control of radiopharmaceutical preparations, brings an additional security to the patients for whom these products are destined [fr

  5. Platelet Preparations for Use in Facial Rejuvenation and Wound Healing: A Critical Review of Current Literature.

    Science.gov (United States)

    Sclafani, Anthony P; Azzi, James

    2015-08-01

    In facial plastic surgery, the potential for direct delivery of growth factors from platelet preparations has been of particular interest for use in facial rejuvenation, recovery after facial surgery, and wound healing. A literature search was conducted through PubMed for the terms PRP, PRFM, platelet-rich plasma, platelet-rich fibrin matrix, platelet preparations, platelet therapy, growth factors, platelet facial, platelet facial rejuvenation, platelet wound healing, platelet plastic surgery. Articles pertaining to the use of platelet preparations in facial surgery and wound healing in plastic surgery after 2001 were included. Thirteen in vitro studies showed use of platelet-rich plasma (PRP) and platelet-rich fibrin matrix (PRFM) had a significant effect on cellular activity. Twenty-four out of 28 animal studies exhibited favorable results with use of a platelet preparation, including five of six studies that showed enhanced fat graft survival with addition of a platelet preparation. Twenty-three case series and clinical trials were identified, only two of which showed no differences. Twenty-one reported favorable results with use of various platelet preparations. A total of 47 studies used PRP, four studies evaluated Leukocyte-rich PRP, and fourteen studies used PRFM. The vast majority of studies examined show a significant and measurable effect on cellular changes, wound healing, and facial esthetic outcomes with use of platelet preparations, both topical and injectable. One must also consider possible publication bias against null results that may have had an influence on the data that were available for review. However, the preponderance of studies suggests that platelet preparations might represent an as-of-yet untapped adjunct in facial plastic surgery.

  6. Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate

    Science.gov (United States)

    Wagner, Eric R.; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M.; Chase, Steven; Westendorf, Jennifer J.; Dietz, Allan B.; van Wijnen, Andre J.; Yaszemski, Michael J.

    2015-01-01

    Purpose: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer “neoligament” seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell–cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). Results: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts. Summary: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration. PMID:26413793

  7. Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

    Science.gov (United States)

    Hildner, F; Eder, M J; Hofer, K; Aberl, J; Redl, H; van Griensven, M; Gabriel, C; Peterbauer-Scherb, A

    2015-07-01

    Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

  8. High-resolution molecular validation of self-renewal and spontaneous differentiation in adipose-tissue derived human mesenchymal stem cells cultured in human platelet lysate

    Science.gov (United States)

    Dudakovic, Amel Dudakovic; Camilleri, Emily; Riester, Scott M.; Lewallen, Eric A.; Kvasha, Sergiy; Chen, Xiaoyue; Radel, Darcie J.; Anderson, Jarett M.; Nair, Asha A.; Evans, Jared M.; Krych, Aaron J.; Smith, Jay; Deyle, David R.; Stein, Janet L.; Stein, Gary S.; Im, Hee-Jeong; Cool, Simon M.; Westendorf, Jennifer J.; Kakar, Sanjeev; Dietz, Allan B.; van Wijnen, Andre J.

    2014-01-01

    Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1 and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10 fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while up-regulating WNT-related genes (WISP2, SFRP2 and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility. PMID:24905804

  9. Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate.

    Science.gov (United States)

    Haack-Sørensen, Mandana; Juhl, Morten; Follin, Bjarke; Harary Søndergaard, Rebekka; Kirchhoff, Maria; Kastrup, Jens; Ekblond, Annette

    2018-04-17

    In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95 × 10 6 cells were suspended in 10% FBS or 5% hPL medium, and loaded into a bioreactor coated with cryoprecipitate. ASCs (P0) were harvested, and 30 × 10 6 ASCs were reloaded for continued expansion (P1). Feeding rate and time of harvest was guided by metabolic monitoring. Viability, sterility, purity, differentiation capacity, and genomic stability of ASCs P1 were determined. Cultivation of SVF in hPL medium for in average nine days, yielded 546 × 10 6 ASCs compared to 111 × 10 6 ASCs, after 17 days in FBS medium. ASCs P1 yields were in average 605 × 10 6 ASCs (PD [population doublings]: 4.65) after six days in hPL medium, compared to 119 × 10 6 ASCs (PD: 2.45) in FBS medium, after 21 days. ASCs fulfilled ISCT criteria and demonstrated genomic stability and sterility. The use of hPL as a growth supplement for ASCs expansion in the quantum cell expansion system provides an efficient expansion process compared to the use of FBS, while maintaining cell quality appropriate for clinical use. The described process is an obvious choice for manufacturing of large-scale allogeneic ASC products.

  10. Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells.

    Science.gov (United States)

    Hofbauer, Pablo; Riedl, Sabrina; Witzeneder, Karin; Hildner, Florian; Wolbank, Susanne; Groeger, Marion; Gabriel, Christian; Redl, Heinz; Holnthoner, Wolfgang

    2014-09-01

    As angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells. The usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs). Expression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities. The use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

    Science.gov (United States)

    Castiglia, Sara; Mareschi, Katia; Labanca, Luciana; Lucania, Graziella; Leone, Marco; Sanavio, Fiorella; Castello, Laura; Rustichelli, Deborah; Signorino, Elena; Gunetti, Monica; Bergallo, Massimiliano; Bordiga, Anna Maria; Ferrero, Ivana; Fagioli, Franca

    2014-06-01

    Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  13. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  14. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture; Cultivo e irradiacao de fibroblastos humanos em meio enriquecido com lisado de plaquetas para obtencao de camada de sustentacao em culturas de celulas da epiderme

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele

    2011-07-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  15. Comparison of different procedures to prepare platelet-rich plasma for studies of platelet aggregation by light transmission aggregometry.

    Science.gov (United States)

    Femia, Eti Alessandra; Pugliano, Mariateresa; Podda, Gianmarco; Cattaneo, Marco

    2012-01-01

    Light transmission aggregometry (LTA), the gold standard for the study of patients with defects of platelet function, is a poorly standardized technique. The guidelines that have been produced so far are largely based on consensus of experts, due to the absence of studies directly comparing different procedures. Therefore, ad hoc studies are needed to gather scientific evidence on how to choose the most appropriate procedures for LTA measurement. In this study, we aimed at evaluating the most appropriate conditions for preparing samples of platelet-rich plasma (PRP) for studies of platelet aggregation by LTA. Citrate-anticoagulated blood from 32 individuals was centrifuged at 150, 200, 250 or 300×g at room temperature for 10 min. Red blood cells contamination was highest in PRP prepared at 150×g; mean platelet volume (MPV) was lowest in PRP prepared at 300×g. The extent of platelet aggregation measured by LTA was lower and more variable in PRP prepared at 300×g. Therefore, centrifugation of blood at 200×g or 250×g for 10 min appears to be the best condition for preparing PRP for LTA studies.

  16. Platelet-rich plasma differs according to preparation method and human variability.

    Science.gov (United States)

    Mazzocca, Augustus D; McCarthy, Mary Beth R; Chowaniec, David M; Cote, Mark P; Romeo, Anthony A; Bradley, James P; Arciero, Robert A; Beitzel, Knut

    2012-02-15

    Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma. Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated. The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed. Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in

  17. Animal models to assess the therapeutic efficacy of human serum and serum-converted platelet lysates for dry eye syndrome: Seeing is believing.

    Science.gov (United States)

    Tseng, Ching-Li; Seghatchian, Jerard; Burnouf, Thierry

    2015-08-01

    There is much interest in the clinical use of serum-converted human blood or platelet concentrates in regenerative medicine, most specifically for wound healing and tissue repair of soft and hard tissues. The scientific rationale supporting the clinical efficacy of these preparations is based on the expectation that their physiological mixture of natural growth factors can orchestrate cell expansion and differentiation in vivo. However, a lack of standardization and regulatory oversight of these blood materials maintain a perception of uncertainty in the scientific and medical community on the value of these preparations for some clinical indications. More studies are needed to understand the mechanism of action underlying their expected efficacy and standardize their use, and benefit from their biological versatility. One application of serum is as eye drop for treating dry eye syndrome (DES), a multifactorial disease of the ocular surface, which has a prevalence of 15% of more in the population. DES can lead to chronic inflammation of the ocular surface, surface impairment in the cornea and conjunctiva, and, in patients with Sjogren syndrome, result in a disruption of the ocular surface epithelium. Objective experimental assessment of safety and efficacy of serum eye drops can help establish scientific rationale in optimal product composition and use. This can be achieved, first, through cell cultures with relevant cell models, before considering, then, animal studies using DES animal models. Several models have been evaluated and are reported in this concise review. The model we have developed encompasses the use of rabbits, where their eyes are treated with 0.1% benzalkonium chloride (BAC), a common preservative in ophthalmic agents, 3 times daily for 4 weeks. This relatively mild treatment results in moderate DES pathology, with a stable shortage of tear secretion throughout a 7-week study period, which we found suitable for assessing efficacy of serum eye

  18. White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods

    NARCIS (Netherlands)

    Dijkstra-Tiekstra, M. J.; van der Schoot, C. E.; Pietersz, R. N. I.; Reesink, H. W.

    2005-01-01

    BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments in platelet concentrates (PCs) may induce allo-immunization in the recipient. MATERIALS AND METHODS: As the level of WBC fragments can differ between PCs produced using different methods, we compared PCs prepared by using the buffy-coat

  19. A Comparison of Platelet Count and Enrichment Percentages in the Platelet Rich Plasma (PRP) Obtained Following Preparation by Three Different Methods.

    Science.gov (United States)

    Sabarish, Ram; Lavu, Vamsi; Rao, Suresh Ranga

    2015-02-01

    Platelet rich plasma (PRP) represents an easily accessible and rich source of autologous growth factors. Different manual methods for the preparation of PRP have been suggested. Lacuna in knowledge exists about the efficacy of PRP preparation by these different manual methods. This study was performed to determine the effects of centrifugation rate revolutions per minute (RPM) and time on the platelet count and enrichment percentages in the concentrates obtained following the three different manual methods of PRP preparation. In vitro experimental study. This was an experimental study in which platelet concentration was assessed in the PRP prepared by three different protocols as suggested by Marx R (method 1), Okuda K (method 2) and Landesberg R (method 3). A total of 60 peripheral blood samples, (n=20 per method) were obtained from healthy volunteers. Baseline platelet count was assessed for all the subjects following which PRP was prepared. The platelet count in the PRP was determined using coulter counter (Sysmex XT 2000i). The mean of the platelet count obtained and their enrichment percentage were calculated and intergroup comparison was done (Tukey's HSD test). The number of platelets and enrichment percentage in PRP prepared by method 1 was higher compared to method 2 and method 3; this difference in platelet concentrates was found to be statistically significant (p < 0.05). The centrifugation rate and time appear to be important parameters, which influence the platelet yield. Method 1 which had lower centrifugation rate and time yielded a greater platelet count and enrichment percentage.

  20. Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma.

    Science.gov (United States)

    Shin, Hyeok-Soo; Woo, Heung-Myong; Kang, Byung-Jae

    2017-06-26

    Platelet-rich plasma (PRP) has been expected for regenerative medicine because of its growth factors. However, there is considerable variability in the recovery and yield of platelets and the concentration of growth factors in PRP preparations. The aim of this study was to identify optimal relative centrifugal force and spin time for the preparation of PRP from canine blood using a double-centrifugation tube method. Whole blood samples were collected in citrate blood collection tubes from 12 healthy beagles. For the first centrifugation step, 10 different run conditions were compared to determine which condition produced optimal recovery of platelets. Once the optimal condition was identified, platelet-containing plasma prepared using that condition was subjected to a second centrifugation to pellet platelets. For the second centrifugation, 12 different run conditions were compared to identify the centrifugal force and spin time to produce maximal pellet recovery and concentration increase. Growth factor levels were estimated by using ELISA to measure platelet-derived growth factor-BB (PDGF-BB) concentrations in optimised CaCl 2 -activated platelet fractions. The highest platelet recovery rate and yield were obtained by first centrifuging whole blood at 1000 g for 5 min and then centrifuging the recovered platelet-enriched plasma at 1500 g for 15 min. This protocol recovered 80% of platelets from whole blood and increased platelet concentration six-fold and produced the highest concentration of PDGF-BB in activated fractions. We have described an optimised double-centrifugation tube method for the preparation of PRP from canine blood. This optimised method does not require particularly expensive equipment or high technical ability and can readily be carried out in a veterinary clinical setting.

  1. An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

    Directory of Open Access Journals (Sweden)

    Taisuke Watanabe

    2017-01-01

    Full Text Available Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF and concentrated growth factors (CGF according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately.

  2. Effect of Titanium-prepared Platelet-rich Fibrin Treatment on the ...

    African Journals Online (AJOL)

    Effect of Titanium-prepared Platelet-rich Fibrin Treatment on the Angiogenic Biomarkers in Gingival Crevicular Fluid in Infrabony Defects of Patients with Chronic Periodontitis: A Randomized Controlled Clinical Trial.

  3. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

    Science.gov (United States)

    2013-01-01

    Introduction Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. Methods PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Results Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of

  4. Quantification of platelets and platelet derived growth factors from platelet-rich-plasma (PRP) prepared at different centrifugal force (g) and time.

    Science.gov (United States)

    Arora, Satyam; Doda, Veena; Kotwal, Urvershi; Dogra, Mitu

    2016-02-01

    Platelet derived biomaterials represent a key source of cytokines and growth factors extensively used for tissue regeneration; wound healing and tissue repair. Our study was to quantify platelets and growth factors released by PRP when prepared at different centrifugal force (g) and time. Our study was approved by the institutional ethical committee. One hundred millilitres of whole blood (WB) was collected in bag with CPDA as the anticoagulant(AC); (14 mL for 100 mL WB ratio). Nine aliquots of 10 mL each were made from the bag and set of three aliquots were made a group. PRP was prepared at varying centrifugal force (group A: -110 g, group B: -208 g & group C: -440 g) & time (1: -5 min, 2: -10 min & 3: -20 min). Contents of each PRP prepared were analysed. Commercial sandwich ELISA kits were used to quantify the concentrations of CD62P (Diaclone SAS; France), Platelet derived growth factors-AB (Qayee-Bio; China), transforming growth factor-β1 (DRG; Germany) and vascular endothelial growth factor (Boster Immuno Leader; USA) released in each PRP prepared. Eight volunteers were enrolled in the study (24-30 years). The baseline blood counts of all the volunteers were comparable (p ≥ 0.05). Mean ± SD of platelet yield of all nine groups ranged from 17.2 ± 4.2% to 78.7 ± 5.7%. Each PRP was activated with calcified thromboplastin to quantify the growth factors released by them. Significantly higher (p < 0.05) transforming growth factor-β1 and vascular endothelial growth factor were released compared to the baseline. Our study highlights the variation in both force (g) and time results in changes at cellular level and growth factor concentrations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors.

    Science.gov (United States)

    Amable, Paola Romina; Carias, Rosana Bizon Vieira; Teixeira, Marcus Vinicius Telles; da Cruz Pacheco, Italo; Corrêa do Amaral, Ronaldo José Farias; Granjeiro, José Mauro; Borojevic, Radovan

    2013-06-07

    Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 10(6) to 1.9 × 10(6) platelets/μl). Platelets were highly purified, because only platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF. Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool

  6. Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

    Science.gov (United States)

    Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

    2015-09-09

    Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of

  7. Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation.

    Science.gov (United States)

    Shen, E-Chin; Chou, Tz-Chong; Gau, Ching-Hwa; Tu, Hsiao-Pei; Chen, Yen-Teen; Fu, Earl

    2006-10-01

    Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.

  8. Indium-111 tropolone, a new high-affinity platelet label: preparation and evaluation of labeling parameters

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Rao, S.A.; Didisheim, P.

    1981-01-01

    Platelets were isolated with a new neutral, lipid-soluble metal complex of indium-111 and tropolone. Unlike oxine, which must be dissolved in ethyl alcohol, tropolone is soluble saline. Platelet labeling with In-111 tropolone can be performed in both acid-citrate-dextrose (ACD)-plasma and ACD-saline media within two hours' time. Labeling efficiency has been 80-90% in ACD-saline and 60-70% in the ACD-plasma medium. Optimum concentrations for the labeling of platelets with In-111 tropolone were 5 micrograms/ml in ACD-saline and 10 micrograms/ml in ACD-plasma, using a 15-min incubation at room temperature. A kit formulation for convenient routine preparation of In-111-labeled platelets has been developed. Seven parameters of platelet labeling were studied: concentration of tropolone, citrate, plasma proteins, and calcium ions; also platelet density, temperature, and pH of incubation medium. Their effects on the mechanism of platelet labeling with lipid-soluble tracers are discussed

  9. In vivo evaluation of titanium-prepared platelet-rich fibrin (T-PRF): a new platelet concentrate.

    Science.gov (United States)

    Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Fıratlı, Erhan

    2013-07-01

    We have developed a new, titanium-prepared, platelet-rich fibrin (T-PRF) together with the protocol for forming it, which is based on the hypothesis that titanium tubes may be more effective at activating platelets than the glass tubes used by Chouckroun in his platelet-rich fibrin (PRF) method. The aim of this study was to find a suitable animal model in which to evaluate the method and to investigate the efficacy of T-PRF for wound healing. Blood samples from 6 rabbits were used to confirm the protocol for formation of T-PRF. We evaluated T-PRF or T-PRF-like clots morphologically using scanning electron microscopy (EM). Blood samples from 5 rabbits were used to develop an experiment in which to evaluate the effects of T-PRF on wound healing. The mucoperiosteal flaps were filled with autologous T-PRF membranes from the vestibule in the anterior mandibular regions. Samples collected from the surgical sites were stained with haematoxylin and eosin. We found a mature fibrin network in T-PRF clots that had been centrifuged for 15 min at 3500 rpm and, 15 days after placement of the membrane, we found newly-forming connective tissue and islets of bony tissue in the T-PRF membrane. These results show that T-PRF could induce the formation of new bone with new connective tissue in a rabbit model of wound healing within 30 days of treatment. Published by Elsevier Ltd.

  10. Distribution, recovery and concentration of platelets and leukocytes in L-PRP prepared by centrifugation.

    Science.gov (United States)

    de Melo, Bruna Alice Gomes; Martins Shimojo, Andréa Arruda; Marcelino Perez, Amanda Gomes; Duarte Lana, José Fabio Santos; Andrade Santana, Maria Helena

    2018-01-01

    Platelet-rich plasma (PRP) is an autologous product prepared from whole blood (WB) that is widely used in regenerative medicine. In clinical practice, discontinuous centrifugation is used for both hand- and machine-prepared PRP. However, separation of WB fractions via centrifugation is a complex process, and the lack of clear mechanisms limits the understanding and evaluation of PRP preparation methods This paper focuses on the distribution, recovery and concentration factor of platelets and leukocytes in L-PRP (leukocyte and platelet-rich plasma) to define a concentration pattern for these blood components due to centrifugation conditions. WB collected from three healthy donors was centrifuged for 10min at 50-800 xg in a first step and then at 400 xg in a second step. The results from the first centrifugation step showed most platelets to be distributed in the upper layer (UL) and the buffy coat (BC), with approximately 14.5±5.2% retained in the bottom layer (BL). Most leukocytes were present in the BL. The greatest platelet recoveries from L-PRP were obtained at up to 150 xg (88.5±16.9%). The cumulative concentration factors with respect to the WB from the second centrifugation step were 6 and 1.2 for platelets and leukocytes, respectively. Thus, the concentration patterns delineated three centrifugation ranges with platelet/leukocyte ratios of 205±18, 325±15 and 107±4 and lymphocyte/granulocyte ratios of 1.54±0.74, 0.90±0.08 and 0.42±0.07. These findings contribute to a scientifically based standardization of L-PRP preparations. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. [Quality of buffy-coat-derived platelet concentrates prepared using automated system terumo automated centrifuge and separator integration (TACSI)].

    Science.gov (United States)

    Zebrowska, Agnieszka; Lipska, Alina; Rogowska, Anna; Bujno, Magdalena; Nedzi, Marta; Radziwon, Piotr

    2011-03-01

    Platelet recovery, and viability, and function is strongly dependent on the method of the preparation of platelet concentrate (PC). The glucose consumption, decrease of pH, release of alpha granules during storage in platelet concentrate impair their clinical effectiveness. To compare of the quality of buffy-coat-derieved platelet concentrates prepared using automatic system terumo automated centrifuge and separator integration (TACSI) and stored over 7 days. PCs were prepared from buffy coats using manual method (group I), or automatic system TACSI (group II). Fifteen PCs prepared from the 5 buffy coats each were stored over 7 days in 22-24 degrees C and tested. Samples were taken from the PCs container on days 1 and 7. The following laboratory tests were performed: number of platelets, platelets derived microparticles, CD62P expression, platelet adhesion, pH, glucose, lactate dehydrogenase activity. We have observed higher expression of CD62P in PCs prepared using manual method compared to the PCs produced automatically Platelet recovery was significantly higher in PCs prepared using automatic systems compare to manual method. Compared to manual methods, automatic system for preparation of buffy coats, is more efficient and enable production of platelets concentrates of higher quality.

  12. Preparing Platelet-Rich Plasma with Whole Blood Harvested Intraoperatively During Spinal Fusion.

    Science.gov (United States)

    Shen, Bin; Zhang, Zheng; Zhou, Ning-Feng; Huang, Yu-Feng; Bao, Yu-Jie; Wu, De-Sheng; Zhang, Ya-Dong

    2017-07-22

    BACKGROUND Platelet-rich plasma (PRP) has gained growing popularity in use in spinal fusion procedures in the last decade. Substantial intraoperative blood loss is frequently accompanied with spinal fusion, and it is unknown whether blood harvested intraoperatively qualifies for PRP preparation. MATERIAL AND METHODS Whole blood was harvested intraoperatively and venous blood was collected by venipuncture. Then, we investigated the platelet concentrations in whole blood and PRP, the concentration of growth factors in PRP, and the effects of PRP on the proliferation and viability of human bone marrow-derived mesenchymal stem cells (HBMSCs). RESULTS Our results revealed that intraoperatively harvested whole blood and whole blood collected by venipuncture were similar in platelet concentration. In addition, PRP formulations prepared from both kinds of whole blood were similar in concentration of platelet and growth factors. Additional analysis showed that the similar concentrations of growth factors resulted from the similar platelet concentrations of whole blood and PRP between the two groups. Moreover, these two kinds of PRP formulations had similar effects on promoting cell proliferation and enhancing cell viability. CONCLUSIONS Therefore, intraoperatively harvested whole blood may be a potential option for preparing PRP spinal fusion.

  13. Preparation of leukocyte-poor platelet concentrates from buffy coats. I. Special inserts for centrifuge cups

    NARCIS (Netherlands)

    Pietersz, R. N.; Reesink, H. W.; Dekker, W. J.; Fijen, F. J.

    1987-01-01

    A special insert was developed for centrifuge cups in order to prepare leukocyte-poor platelet concentrates from buffy coats by using quadruple citrate phosphate dextrose-saline adenine glucose mannitol systems from different manufacturers. Each centrifuge cup could contain up to 4 sets of double

  14. Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood

    Science.gov (United States)

    Rebulla, Paolo; Pupella, Simonetta; Santodirocco, Michele; Greppi, Noemi; Villanova, Ida; Buzzi, Marina; De Fazio, Nicola; Grazzini, Giuliano

    2016-01-01

    Background In addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel. Materials and methods Cord blood units collected at public banks with total nucleated cell counts 150×109/L and volume >50 mL, underwent soft centrifugation within 48 hours of collection. Platelet-rich plasma was centrifuged at high speed to obtain a CBPC with target platelet concentration of 800–1,200×109/L, which was cryopreserved, without cryoprotectant, below −40 °C. Results During 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×109/L. Total platelet count per CBPC was 11.3±4.9×109. Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC. Discussion This study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC. PMID:26509822

  15. Use of Platelet Preparations in Facial Rejuvenation and Wound Healing Remains Unproven.

    Science.gov (United States)

    Kenworthy, William; Langridge, Benjamin; Patel, Nakul; Waterhouse, Norman

    2016-04-01

    There is growing interest in the use of platelet derivatives in facial aesthetic surgery and wound healing. Sclafani et al. have concluded from their review of the literature that the vast majority of studies show a significant and measurable effect on facial aesthetic outcomes with the use of these platelet preparations. We suggest that an alternative review of the same literature may well have produced a different set of conclusions. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

  16. Pooled human platelet lysate versus fetal bovine serum-investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use.

    Science.gov (United States)

    Trojahn Kølle, Stig-Frederik; Oliveri, Roberto S; Glovinski, Peter V; Kirchhoff, Maria; Mathiasen, Anders Bruun; Elberg, Jens Jørgen; Andersen, Peter Stemann; Drzewiecki, Krzysztof Tadeusz; Fischer-Nielsen, Anne

    2013-09-01

    Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS)). ASCs from four healthy donors were cultured in either DMEM(pHPL) or DMEM(FBS), and the population doubling time (PDT) was calculated. ASCs from two of the donors were expanded in DMEM(pHPL) or DMEM(FBS) and cultured for the final week before harvesting with or without the addition of vascular endothelial growth factor. We assessed the chromosomal stability (through the use of array comparative genomic hybridization), the expression of ASC and endothelial surface markers and the differentiation and angiogenic potential of these cells. The ASCs that were cultured in pHPL exhibited a significantly shorter PDT of 29.6 h (95% confidence interval, 22.3-41.9 h) compared with those cultured in FBS, for which the PDT was 123.9 h (95% confidence interval, 95.6-176.2 h). Comparative genomic hybridization analyses revealed no chromosomal aberrations. Cell differentiation, capillary structure formation and cell-surface marker expression were generally unaffected by the type of medium supplement that was used or by the addition of vascular endothelial growth factor. We observed that the use of pHPL as a growth supplement for ASCs facilitated a significantly higher proliferation rate compared with FBS without compromising genomic stability or differentiation capacity. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  17. [Assessment study on a set of platelet-rich plasma preparation].

    Science.gov (United States)

    Li, Ming; Zhang, Changqing; Yuan, Ting; Chen, Shengbao; Lü, Ruju

    2011-01-01

    To calculate the recovery rate and enrichment factor and to analyse the correlation by measuring the concentrations of platelets, leukocyte, and growth factors in platelet-rich plasma (PRP) so as to evaluate the feasibility and stability of a set of PRP preparation. The peripheral blood (40 mL) was collected from 30 volunteers accorded with the inclusion criteria, and then 4 mL PRP was prepared using the package produced by Shandong Weigao Group Medical Polymer Company Limited. Automatic hematology analyzer was used to count the concentrations of platelets and leukocyte in whole blood and PRP. The enrichment factor and recovery rate of platelets or leukocyte were calculated; the platelet and leukocyte concentrations of male and female volunteers were measured, respectively. The concentrations of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), and vascular endothelial growth factor (VEGF) were assayed by ELISA. The platelet concentrations of whole blood and PRP were (131.40 +/- 29.44) x 10(9)/L and (819.47 +/- 136.32) x 10(9)/L, respectively, showing significant difference (t = 27.020, P = 0.000). The recovery rate of platelets was 60.85% +/- 8.97%, and the enrichment factor was 6.40 +/- 1.06. The leukocyte concentrations of whole blood and PRP were (5.57 +/- 1.91) x 10(12)/L and (32.20 +/- 10.42) x 10(12)/L, respectively, showing significant difference (t = 13.780, P = 0.000). The recovery rate of leukocyte was 58.30% +/- 19.24%, and the enrichment factor was 6.10 +/- 1.93. The concentrations of platelets and leukocyte in PRP were positively correlated with the platelet concentration (r = 0.652, P = 0.000) and leukocyte concentration (r = 0.460, P = 0.011) in whole blood. The concentrations of platelet and leukocyte in PRP between male and female were not significantly different (P > 0.05). The concentrations of PDGF, TGF-beta, and VEGF in PRP were (698.15 +/- 64.48), (681.36 +/- 65.90), and (1071.55 +/- 106.04) ng/mL, which were

  18. Effect of two different preparations of platelet-rich plasma on synoviocytes.

    Science.gov (United States)

    Assirelli, Elisa; Filardo, Giuseppe; Mariani, Erminia; Kon, Elizaveta; Roffi, Alice; Vaccaro, Franca; Marcacci, Maurilio; Facchini, Andrea; Pulsatelli, Lia

    2015-09-01

    To analyse the modifications induced by two different platelet-rich plasma (PRP) preparations on osteoarthritis (OA) synoviocytes, by documenting changes in gene expression of factors involved in joint physiopathology. OA synoviocytes were cultured for 7 days in medium with different concentrations of either P-PRP (a pure platelet concentrate without leucocytes but with a limited number of platelets), L-PRP (a higher platelet concentrate containing leucocytes) or platelet-poor plasma (PPP). Gene expression of interleukin (IL)-1beta, IL-6, IL-8/CXCL8, tumour necrosis factor alpha, IL-10, IL-4, IL-13, metalloproteinase-13, tissue inhibitor of metalloproteinase (TIMP)-1, (TIMP)-3, (TIMP)-4, vascular endothelial growth factor, transforming growth factor beta1, fibroblast growth factor (FGF)-2, hepatocyte growth factor (HGF), hyaluronic acid (HA) synthases (HAS)-1, (HAS)-2, and (HAS)-3 was analysed by RT-PCR. HA production was determined in culture supernatants by ELISA. IL-1β, IL-8 and FGF-2 were significantly induced by L-PRP compared to both P-PRP and PPP; HGF was down-modulated by L-PRP versus both P-PRP and PPP, and an inverse dose-response influence was shown for all preparations. Expression level of TIMP-4 was lower in the presence of L-PRP compared with P-PRP. HA production and HAS gene expression did not seem to be modulated by PRP. L-PRP is able to sustain the up-regulation of proinflammatory factors, (IL-1beta, IL-8 and FGF-2), together with a down-modulation of HGF and TIMP-4 expression, two factors that have been recognized as anti-catabolic mediators in cartilage, thus supporting the need to further optimize the PRP preparations to be applied in clinical practice.

  19. Platelet-rich fibrin prepared from stored whole-blood samples.

    Science.gov (United States)

    Isobe, Kazushige; Suzuki, Masashi; Watanabe, Taisuke; Kitamura, Yutaka; Suzuki, Taiji; Kawabata, Hideo; Nakamura, Masayuki; Okudera, Toshimitsu; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh; Tanaka, Takaaki; Kawase, Tomoyuki

    2017-12-01

    In regenerative therapy, self-clotted platelet concentrates, such as platelet-rich fibrin (PRF), are generally prepared on-site and are immediately used for treatment. If blood samples or prepared clots can be preserved for several days, their clinical applicability will expand. Here, we prepared PRF from stored whole-blood samples and examined their characteristics. Blood samples were collected from non-smoking, healthy male donors (aged 27-67 years, N = 6), and PRF clots were prepared immediately or after storage for 1-2 days. Fibrin fiber was examined by scanning electron microscopy. Bioactivity was evaluated by means of a bioassay system involving human periosteal cells, whereas PDGF-BB concentrations were determined by an enzyme-linked immunosorbent assay. Addition of optimal amounts of a 10% CaCl 2 solution restored the coagulative ability of whole-blood samples that contained an anticoagulant (acid citrate dextrose) and were stored for up to 2 days at ambient temperature. In PRF clots prepared from the stored whole-blood samples, the thickness and cross-links of fibrin fibers were almost identical to those of freshly prepared PRF clots. PDGF-BB concentrations in the PRF extract were significantly lower in stored whole-blood samples than in fresh samples; however, both extracts had similar stimulatory effects on periosteal-cell proliferation. Quality of PRF clots prepared from stored whole-blood samples is not reduced significantly and can be ensured for use in regenerative therapy. Therefore, the proposed method enables a more flexible treatment schedule and choice of a more suitable platelet concentrate immediately before treatment, not after blood collection.

  20. Comparing different preparation methods to study human fibrin fibers and platelets using TEM.

    Science.gov (United States)

    Buys, Antoinette V; Pretorius, Etheresia

    2012-06-01

    For the study of cellular ultrastructure, the sample needs to be stabilized by fixation, with the ultimate aim to preserve the native tissue organization and to protect the tissue against later stages of preparation. Chemical and freezing fixation are most used, and chemical fixation employs agents that permeate tissues and cells by diffusion and covalently bind with their major biochemical constituents to fix them. Most widely used chemical fixatives are aldehydes, e.g., formaldehyde and glutaraldehyde, which are noncoagulating, crosslinking agents. Cryofixation methods for ultrastructural studies are also popular, and high-pressure freezing immobilizes all cell constituents and arrests biological activity by removing the thermal energy from the system. In the current research, we used platelet-rich plasma (PRP) to study expansive fibrin fibers and platelet ultrastructure to compare the two fixation techniques. We also used thrombin and calcium chloride as a clotting agent to determine the technique most suitable for the formation of extensive fibrin networks. Chemically fixated fibrin fibers were more compact and condensed and also showed a banding pattern on longitudinal sections. High-pressure frozen samples were more dispersed while platelets fixated showed better preserved cellular membranes and organelle structure. PRP coagulated by addition of CaCl(2) showed blood platelets that are noticeably more activated compared with PRP; however, with thrombin, a sharp ultrastructure was seen. We conclude that PRP mixed with thrombin, and freeze substituted, is the most suitable method for the study of extensive fibrin fibers as well as platelets. Copyright © 2011 Wiley Periodicals, Inc.

  1. Detection of endotoxins in radiopharmaceutical preparations. II. Comparison of the sensitivity of methods using the rabbit and the Limulus amoebocyte lysate for the detection of endotoxins

    Energy Technology Data Exchange (ETDEWEB)

    Bruneau, J; Cohen, Y; Merlin, L; Peysson, S

    1986-01-01

    The rise of the rabbit internal temperature after i.v. injection of an endotoxin solution is proportional to concentration. Gelation of Limulus amoebocyte, when in presence of an endotoxin solution, is also related to concentration. We compared the sensitivity of these two methods. With our experimental procedure, the rabbit is sensitive to a 0.40 ng/mL solution and the Limulus amoebocyte lysate to a 0.14 ng/mL solution. The rabbit sensitivity increase is related to the per kilogramme injected volume, whereas sensitivity is not related to the volume to check in the case of the lysate.

  2. Elemental composition of platelets. Part I. Sampling and sample preparation of platelets for trace-element analysis

    International Nuclear Information System (INIS)

    Iyengar, G.V.; Borberg, H.; Kasperek, K.; Kiem, J.; Siegers, M.; Feinendegen, L.E.; Gross, R.

    1979-01-01

    Sampling of platelets for trace-element analysis poses special problems: obtaining adequate sample materials, achieving a sufficient cell purity, preserving viability (integrity), correcting for trapped plasma, and controlling contamination. We used a blood-cell separator for the primary isolation of platelets from blood, and differential centrifugation in natural plasma to further isolate them. The pyrimidopyrimidine RA233 was used as a stabilizer to maintain viability. 131 I-labeled human serum albumin was used to estimate trapped plasma. Contamination was controlled by using five-times-distilled water to simulate donor's blood in the system and by comparing three fractions: the serum, the first portion of the platelet-rich plasma, and the supernatant plasma after the final centrifugation. Neutron activation analysis was used for the elemental analysis. A single differential centrifugation of the platelet-rich plasma from the blood-cell separator at 400 x g for 8 min was optimum (mean mass fractions: erythrocytes/platelets < 5 mg/g and leukocytes/platelets < 20 mg/g). The trapped plasma in the wet platelet samples amounted to about 0.40 g/g. No appreciable contamination from the sampling system was found for the elements Ag, Cd, Co, Cr, Cs, Cu, Fe, Mo, Rb, Sb, Se, and Zn. 2 figures, 3 tables

  3. Characteristics of canine platelet-rich plasma prepared with five commercially available systems.

    Science.gov (United States)

    Franklin, Samuel P; Garner, Bridget C; Cook, James L

    2015-09-01

    To characterize platelet-rich plasma (PRP) products obtained from canine blood by use of a variety of commercially available devices. Blood samples from 15 dogs between 18 months and 9 years of age with no concurrent disease, except for osteoarthritis in some dogs. PRP products were produced from blood obtained from each of the 15 dogs by use of each of 5 commercially available PRP-concentrating systems. Complete blood counts were performed on each whole blood sample and PRP product. The degree of platelet, leukocyte, and erythrocyte concentration or reduction for PRP, compared with results for the whole blood sample, was quantified for each dog and summarized for each concentrating system. The various PRP-concentrating systems differed substantially in the amount of blood processed, method of PRP preparation, amount of PRP produced, and platelet, leukocyte, and erythrocyte concentrations or reductions for PRP relative to results for whole blood. The characteristics of PRP products differed considerably. Investigators evaluating the efficacy of PRPs need to specify the characteristics of the product they are assessing. Clinicians should be aware of the data (or lack of data) supporting use of a particular PRP for a specific medical condition.

  4. Defining an appropriate leucoreduction strategy by serial assessment of cytokine levels in platelet concentrates prepared by different methods

    Directory of Open Access Journals (Sweden)

    Daljit Kaur

    2015-01-01

    Full Text Available Background and Objectives: Different methods of platelet concentrate preparations leave behind certain number of residual leukocytes, accounting for most of the febrile nonhemolytic transfusion reactions, especially in multitransfused patients. Various inflammatory cytokines, such as tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, and IL-6 are generated during storage and have been implicated for these adverse effects. We have studied the levels of these cytokines and their correlation with leucocyte contents in platelet concentrates prepared by three different methods. Study Design and Methods: Five pools of platelet rich plasma platelet concentrates (PRP-PC and buffy-coat platelet concentrates (BC-PC each were prepared and divided into two halves. One half of the pool was leucofiltered (LF, whereas the other half was stored as such. Ten apheresis units were also included in the study. All the platelet concentrates were assessed for leucocyte load and cytokine content (IL-1β, IL-6, and TNF-α on different days of storage (0, 3, and 5 using Nageotte chamber and commercially available immunoassays respectively. Results: There was a statistically significant rise in cytokine levels (IL-1β, IL-6, and TNF-α in nonleucofiltered (NLF random donor platelet concentrates (RDPs (PRP-PC and BC-PC during storage (day 3 and 5 whereas LF RDP concentrates (PRP-PC and BC-PC and apheresis platelet concentrates (AP-PC did not show any significant rise in cytokine levels (on day 3 and 5 over the baseline values at day 0. Conclusion: This data suggests that although AP-PCs are superior to PRP-PC (NLF and BC-PC (NLF in terms of in vitro quality control parameters and cytokine generation during storage, BC-PC mode of platelet preparation followed by leucofiltration is the best method to store platelets and minimise the cytokine accumulation. This strategy is best suited for transfusion in multitransfused hematooncologic patients, who cannot afford

  5. Platelet-rich Preparation may serve as a Powerful Tool for Therapeutic Dental Pulp Regeneration

    Directory of Open Access Journals (Sweden)

    Hai-Hua Sun

    2011-09-01

    Full Text Available Introduction: Regeneration of dental pulp tissues presents one of the most challenging issues in regenerative dentistry due to their extremely poor intrinsic ability for self-healing and re-growth.The hypothesis: We hypothes-ize that patient-derived platelet-rich preparation can be used in clinical endodontic regenerative procedure, serving as a powerful tool for therapeutic dental pulp regeneration.Evaluation of the hypothesis: The cell transplantation does not always obtain the good result because of the low survival rate of transplanted cells. In addition, the use of ex vivo manipulated cell products faces many translational hurdles in treating non-vital disease. Recently, the body cells are focused as a potential source for therapeutics. Some researchers have demonstrated that endogenous stem cells may be recruited to a desired anatomic site pharma-cologically. This is spurring interest in developing new generation of biomaterials that incorporate and release selected powerful extracellular influences in a near-physiological fashion, and subsequently capture endogenous stem cells and influence their fates for regene-ration. The use of patient-derived products such as platelet-rich preparations that contain a multitude of endogenous growth factors and proteins is a clinically translatable biotechnology for this proposes. These simple and cost efficient procedures may have a potential impact in reducing the economic costs for standard medical treatments in regenerative endodontics.

  6. Preparation of a viable population of indium-111-labelled human blood platelets

    International Nuclear Information System (INIS)

    Heyns, A.; Badenhorst, P.N.; Pieters, H.; Loetter, M.G.; Minnaar, P.C.; Duyvene de Wit, L.J.; Reenen, O.R. van; Retief, F.P.; University of the Orange Free State, Bloemfontein; University of the Orange Free State, Bloemfontein; University of the Orange Free State, Bloemfontein

    1980-01-01

    Factors influencing labelling of human platelets with 111 Indium-8-hydroxyquinoline ([ 111 In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22 0 C with [ 111 In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally suspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubationin PPP at 37 0 C for 30 min. The 111 In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies. (orig.) [de

  7. Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

    Science.gov (United States)

    Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian

    2015-10-01

    To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP

  8. Comparison of the platelet-rich plasma and buffy coat protocols for preparation of canine platelet concentrates.

    Science.gov (United States)

    Hoareau, Guillaume L; Jandrey, Karl E; Burges, Julie; Bremer, Daphne; Tablin, Fern

    2014-12-01

    Platelet (PLT) concentrates (PC) can be produced via the buffy coat (BC) or platelet-rich plasma (PRP) protocols. The 2 methods have not been compared with canine blood. The aims of the study were to compare the PLT, WBC, and RBC concentrations, in vitro PLT function, and markers of platelet storage lesion (PSL) in canine PC generated by 2 different protocols, and determine microbial growth throughout storage. PC from 8 healthy donor dogs were produced using 2 standard protocols, PRP and BC. PLT, WBC, and RBC counts, optical aggregometry assays, and PSL markers (pH, pCO2 , HCO3 , lactate and glucose concentrations, and LDH activity) were determined on storage days 0, 1, 3, 5, and 7. Aerobic and anaerobic bacterial cultures were also performed. Mean PLT counts were comparable between protocols and remained stable throughout storage up to day 7, while median WBC and RBC counts on day 0 were significantly higher in the BC-PC group (17,800 WBCs/μL; 195,000 RBCs/μL) than in the PRP-PC group (200 WBCs/μL; 10,000 RBCs/μL) (P = .012). In PRP-PC aggregometry, the median slope and amplitude in response to γ-thrombin and convulxin (+ ADP) were significantly decreased, and virtually absent in BC-PC during storage. PSL markers (lactate, LDH activity) were higher in BC-PC. Aerobic bacterial growth was observed in 2 PRP-PC and 1 BC-PC. This in vitro study suggests that PRP-PC had lesser WBC and RBC contamination and superior PLT function compared with BC-PC. In vivo studies are required to address safety and efficacy of PRP-PC. © 2014 American Society for Veterinary Clinical Pathology.

  9. A Systematic Review of Autologous Platelet-Rich Plasma and Fat Graft Preparation Methods.

    Science.gov (United States)

    Luck, Joshua; Smith, Oliver J; Mosahebi, Afshin

    2017-12-01

    The addition of platelet-rich plasma (PRP) to adipose tissue may improve fat graft survival, although graft retention rates vary markedly between studies. To what extent this outcome heterogeneity reflects differing methodological factors remains unknown. This systematic review aims to synthesize and critically review methodological approaches to autologous PRP and fat cotransplantation in both human and animal studies. In accordance with PRISMA guidelines, Ovid MEDLINE, Scopus, and Cochrane Library databases were searched from inception to April 2017. Data were extracted from all in vivo studies involving autologous PRP and fat cotransplantation. A secondary aim was to assess reporting of technical detail; authors were not contacted to provide missing data. From 335 articles, 23 studies were included in the qualitative synthesis. Some 21 were performed in humans and 2 in rabbits. Six studies were randomized control trials; the remainder reported on observational data. Methods of PRP extraction and activation varied markedly between studies. Fat graft preparation was comparatively more consistent. Methods of PRP and fat mixing differed significantly, especially with regards to relative volume/volume ratios. Our study represents the first systematic review of methodological factors in autologous PRP and fat cotransplantation. It demonstrates that technical factors in graft preparation and administration vary significantly between in vivo studies. Such methodological heterogeneity may explain observed differences in experimental and clinical outcomes. Reporting of key procedural information is inconsistent and often inadequate. These issues make meaningful evaluation of the PRP-enhanced fat grafting literature difficult and may limit its translation into clinical practice.

  10. Quality Assessment of Platelet-Rich Fibrin-Like Matrix Prepared from Whole Blood Samples after Extended Storage

    Directory of Open Access Journals (Sweden)

    Hideo Kawabata

    2017-09-01

    Full Text Available The platelet-rich fibrin–like matrix (PRFM is usually prepared onsite and immediately used for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without quality deterioration, we developed a method for preparation of PRFMs from short-term-stored whole blood (WB samples. In this study, to evaluate the practical expiration date of storage, we extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl2 in glass tubes and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth Factor-BB (PDGF-BB levels were examined by scanning electron microscopy (SEM, flow cytometry, and an Enzyme-Linked ImmunoSorbent Assay (ELISA, respectively. Long-term storage had no significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the coagulation activity may diminish, we propose that PRFMs can be prepared—without evident loss of quality—from WB samples stored for up to 7 days by our previously developed method.

  11. Effect of Titanium-prepared Platelet-rich Fibrin Treatment on the ...

    African Journals Online (AJOL)

    2018-02-07

    Feb 7, 2018 ... Defects of Patients with Chronic Periodontitis: A Randomized. Controlled ... Faculty of Dentistry Department of Periodontology, Kirikkale. University ..... using leukocyte and platelet-rich fibrin: A report of a case. Case. Rep Dent ...

  12. Preparation of leukodepleted platelet concentrates from pooled buffy coats: prestorage filtration with Autostop BC

    NARCIS (Netherlands)

    Pietersz, R. N.; van der Meer, P. F.; Steneker, I.; Hinloopen, B.; Dekker, W. J.; van Zanten, A. P.; Reesink, H. W.

    1999-01-01

    BACKGROUND AND OBJECTIVES: Our requirements for leukocyte-depleted platelet concentrates (LD-PC) for an adult patient are: platelets >240x10(9), leukocytes <5x10(6), volume of 150-400 ml; and at the end of storage a pH between 6.8 and 7.4 and presence of the swirling effect. Our aim was to develop a

  13. Theoretical prediction and validation of cell recovery rates in preparing platelet-rich plasma through a centrifugation.

    Science.gov (United States)

    Piao, Linfeng; Park, Hyungmin; Jo, Chris Hyunchul

    2017-01-01

    In the present study, we propose a theoretical framework to predict the recovery rates of platelets and white blood cells in the process of centrifugal separation of whole blood contained in a tube for the preparation of platelet-rich plasma. Compared to previous efforts to optimize or standardize the protocols of centrifugation, we try to further the physical background (i.e., based on the multiphase flow phenomena) of analysis to develop a universal approach that can be applied to widely different conditions. That is, one-dimensional quasi-linear partial differential equation to describe the centrifugal sedimentation of dispersed phase (red and white blood cells) in continuous phase (plasma) is derived based on the kinematic-wave theory. With the information of whole blood volume and tube geometry considered, it is possible to determine the positions of interfaces between supernatant/suspension and suspension/sediment, i.e., the particle concentration gradient in a tube, for a wide range of centrifugation parameters (time and acceleration). While establishing a theory to predict the recovery rates of the platelet and white blood cell from the pre-determined interface positions, we also propose a new correlation model between the recovery rates of plasma and platelets, which is found to be a function of the whole blood volume, centrifugal time and acceleration, and tube geometry. The present predictions for optimal condition show good agreements with available human clinical data, obtained from different conditions, indicating the universal applicability of our method. Furthermore, the dependence of recovery rates on centrifugal conditions reveals that there exist a different critical acceleration and time for the maximum recovery rate of platelets and white blood cells, respectively. The other parameters such as hematocrit, whole blood volume and tube geometry are also found to strongly affect the maximum recovery rates of blood cells, and finally, as a strategy

  14. Blood platelet kinetics and platelet transfusion.

    Science.gov (United States)

    Aster, Richard H

    2013-11-01

    The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 prior to centrifugation. We used this approach to characterize platelet kinetics and sites of platelet sequestration in normal and pathologic states and to define the influence of variables such as anticoagulant and ABO incompatibility on post-transfusion platelet recovery. The "acidification" approach enabled much wider use of platelet transfusion therapy until alternative means of producing concentrates suitable for transfusion became available.

  15. [The study of anticoagulants selection in platelet-rich plasma preparation].

    Science.gov (United States)

    Hua, Lei; Lai, Gui; Zhenjun, Liu; Guie, Ma

    2015-07-01

    To investigate the effect of the anticoagulants on PRP quality, so as to clarify the appropriate anticoagulant used in PRP production. The microstructure change of platelets collected via heparin, citrate, acid citrate dextrose (ACD) and citrate-theophylline-adenosine-dipyridamole ( CTAD) was observed by TEM following time course. The extent of spontaneous activation of platelets in four groups was detected by measuring sP-selectin in plasma. The TGF-β1 release amount of activated PRP of four groups was measured. CTAD is superior to other anticoagulants in maintaining the integrity of platelet structures for a long time and preventing platelet spontaneous activation. ACD slightly surpassed heparin and citrate in above two aspects. ACD-PRP and CTAD-PRP released significantly more TGF-β1 compared with heparin and citrate. The PRP quality and biological effects were strongly associated with the type of Anticoagulants. ACD and CTAD are optimal anticoagulants in PRP production for they can maintain platelet viability at a high level.

  16. Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis.

    Science.gov (United States)

    Wahlström, Ola; Linder, Cecilia Halling; Ansell, Anna; Kalén, Anders; Söderström, Mats; Magnusson, Per

    2011-01-01

    Platelets contain numerous growth factors essential for wound and fracture healing. We investigated the gene expression in human osteoblast-like cells stimulated with lysed platelets prepared in acidic, neutral, or alkaline buffers. Lysed platelets prepared in buffers at pH 5.4, 7.4, and 7.9, were added after neutralization to hFOB 1.19 cells. Genome-wide microarray analysis was performed using the Affymetrix GeneChip 7G Scanner. Biometric, cluster, and pathway analyses were performed with GeneSpring GX. Biometric analyses demonstrated that 53 genes were differentially regulated (p ≤ 0.005, ≥2-fold increase). Pathway analysis revealed 10 significant pathways of which eight are common ones regulating bone formation and cancer growth. Eleven genes were selected for quantitative real-time polymerase chain reaction (PCR) based on the microarray analysis of the lysed platelets prepared in the pH 5.4 experiments. In conclusion, acidic preparations of lysed platelet concentrates release factors essential for cell proliferation and particularly cell metabolism under hypoxic conditions. The genetic response from these factors was dominated by genes associated with the same pathways observed in bone formation and cancer growth. Activation of TGF-β in the acidic preparation could be a stimulatory key factor of cell proliferation. These results support the hypothesis that acidification of platelets modifies the stimulatory response of mesenchymal cells in vitro, which is analogous with the observed milieu of a low pH present in wound and fracture sites, as well as in growing tumors.

  17. Blood platelet kinetics and platelet transfusion

    OpenAIRE

    Aster, Richard H.

    2013-01-01

    The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 pr...

  18. Platelet size and age determine platelet function independently

    International Nuclear Information System (INIS)

    Thompson, C.B.; Jakubowski, J.A.; Quinn, P.G.; Deykin, D.; Valeri, C.R.

    1984-01-01

    A study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se-methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms

  19. Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures.

    Science.gov (United States)

    Crowther, M; Ford, I; Jeffrey, R R; Urbaniak, S J; Greaves, M

    2000-10-01

    Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P-selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re-infusion of autologous platelets on platelet function. Autologous platelet-rich plasma (PRP) contained a significantly lower (P platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P-selectin or binding fibrinogen increased significantly after CPB (P platelets responsive to in vitro agonists was decreased (P platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re-infusion of autologous PRP. P-selectin expression had returned to pre-CPB levels 24 h post-operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.

  20. Storage of Buffy-coat-derived platelets in additive solutions: in vitro effects on platelets prepared by the novel TACSI system and stored in plastic containers with different gas permeability.

    Science.gov (United States)

    Sandgren, P; Hild, M; Sjödin, A; Gulliksson, H

    2010-11-01

    The novel TACSI system is designed for automated preparation of platelets (PLTs) from pooled buffy coats (BCs). One TACSI device will handle 6 units at the same time. The aim of our in vitro study is to investigate the effects of using this automated equipment with subsequent storage in two different plastic containers and to compare these results with PLTs prepared by the OrbiSac system. Buffy-coat-derived PLTs (n=8) were prepared by using the TACSI system, including storage in polyvinyl chloride (PVC)-based plastic containers with di, n-decyl phthalate (DnDP) (TACSI R) and BTHC (TACSI T)-based plasticizers. As a reference, the OrbiSac System was used to prepare PLTs (n=8) with subsequent storage in a PVC plastic container with a citrate-based plasticizer (BTHC). In total, 16 TACSI and eight reference units, supplied by approximately 30% plasma and 70% SSP+, were analysed for various in vitro variables during the 7-day storage period. No significant difference in PLT counts, LDH, mean platelet volume (MPV) and adenosine triphosphate between the groups was detected. Glucose was lower (P6·8 (day 7) and swirling remained at the highest level (score=2) for all units throughout storage. Platelets prepared by the TACSI system with subsequent storage in two different PVC-based plastic containers were equivalent to reference PLTs with regard to in vitro characteristics during 7 days of storage. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

  1. Platelets Inhibit Migration of Canine Osteosarcoma Cells.

    Science.gov (United States)

    Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

    2017-01-01

    The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

  2. Platelet concentrates stored in plasma for 72 hours at 22 degrees C prepared from buffycoats of citrate-phosphate-dextrose blood collected in a quadruple-bag saline-adenine-glucose-mannitol system

    NARCIS (Netherlands)

    Pietersz, R. N.; Loos, J. A.; Reesink, H. W.

    1985-01-01

    A method is described to prepare platelet concentrates from the buffycoat of citrate-phosphate-dextrose (CPD) blood in a closed four-bag system and to store the platelets in autologous plasma under sterile conditions. After separation of the blood into plasma, buffycoat and leukocyte- and

  3. Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

    International Nuclear Information System (INIS)

    Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

    2010-01-01

    Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

  4. FUNCTIONALIZATION OF 3D FIBROUS SCAFFOLDS PREPARED USING CENTRIFUGAL SPINNING WITH LIPOSOMES AS A SIMPLE DRUG DELIVERY SYSTEM

    Directory of Open Access Journals (Sweden)

    Michala Rampichová

    2017-06-01

    Full Text Available 3D materials supporting cell adhesion, infiltration and proliferation are crucial for bone tissue engineering. In the current study we combined PCL fibers prepared using centrifugal spinning with adhered liposomes filled with platelet lysate as a natural source of growth factors. The scaffold was seeded with MG-63 cells and tested in vitro as a potential drug delivery system for bone tissue engineering.

  5. Optimization of dendritic cell loading with tumor cell lysates for cancer immunotherapy.

    Science.gov (United States)

    Hatfield, Paul; Merrick, Alison E; West, Emma; O'Donnell, Dearbhaile; Selby, Peter; Vile, Richard; Melcher, Alan A

    2008-09-01

    The immune response to cancer is critically determined by the way in which tumor cells die. As necrotic, stress-associated death can be associated with activation of antitumor immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based vaccination have commonly used freeze-thaw "necrotic" lysates as an immunogenic source of tumor-associated antigens. In this study, the effect of such lysates on the ability of DCs to mature in response to well-established maturation stimuli was examined, and methods to enhance lysate-induced DC activation explored. Freeze-thaw lysates were prepared from murine tumor cell lines and their effects on bone marrow-derived DC maturation and function examined. Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-alpha, and IL-6]. Although IL-10 secretion was increased by lysate-pulsed DCs, this was not responsible for the observed suppression of IL-12. Although activation of the nuclear factor-kappaB pathway remained intact, the kinase activity of phosphorylated p38 mitogen-activated protein kinase was inhibited in lysate-pulsed DCs. Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis, and advocates careful characterization and optimization of tumor cell-derived vaccine sources for cancer immunotherapy.

  6. Radiolabeled platelets

    International Nuclear Information System (INIS)

    Datz, F.L.; Taylor, A.T.

    1986-01-01

    Initial interest in developing techniques to radiolabel platelets was spurred by the lack of an accurate method for measuring platelet life span in both normals and in thrombocytopenic patients. Early investigators could obtain only rough estimates of platelet life spans by monitoring the platelet counts of thrombocytopenic patients undergoing platelet transfusions. Labels were also sought that would allow imaging of platelets in vivo in order to better understand the pathophysiology of atherosclerosis, thrombophlebitis, and clotting disorders, and to improve the clinical diagnosis of these diseases. Two types of platelet labels were investigated: cohort (pulse) labels and random labels. Cohort labels are taken up by megakaryocytes in the bone marrow and incorporated in the DNA and other components of the forming platelet. In theory, only freshly released platelets of a uniform age are labeled. Random labels, on the other hand, tag platelets in the peripheral blood, labeling platelets of all ages

  7. Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

    International Nuclear Information System (INIS)

    Weber, N.; Mangold, H.K.

    1985-01-01

    Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'- 14 C]hexadecyl-sn-glycerol or rac-1-O-[1'- 14 C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'- 14 C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity

  8. Platelet adhesiveness: the effect of centrifugation on the measurement of adhesiveness in platelet-rich plasma

    Science.gov (United States)

    McBride, J. A.

    1968-01-01

    Platelet adhesiveness has been measured in citrated whole blood and in platelet-rich plasma obtained from normal subjects, splenectomized patients, and from patients in whom the diagnosis of recurrent venous thrombosis had been made. The duration of centrifugation used in the preparation of platelet-rich plasma was found to have a profound effect on the measurement of platelet adhesiveness because the figure for platelet adhesiveness measured in platelet-rich plasma obtained by centrifugation was considerably lower than that found in citrated whole blood. This effect was particularly marked when platelet-rich plasma was obtained from subjects in whom platelet adhesiveness measured in whole blood was increased. PMID:5699080

  9. The role of titanium-prepared platelet-rich fibrin in palatal mucosal wound healing and histoconduction.

    Science.gov (United States)

    Ustaoğlu, Gülbahar; Ercan, Esra; Tunali, Mustafa

    2016-10-01

    We aimed to determine the clinical effects of titanium-prepared, platelet-rich fibrin (T-PRF) on human palatal mucosal wound healing (PMWH) and to identify its effect on time-dependent changes in palatal soft-tissue thickness (PSTT) in terms of histoconduction, which is a new concept. Free gingival graft (FGG) donor sites were treated with T-PRF and compared with an untreated control group. The results of colour match and H 2 O 2 -bubbling tests for complete wound epithelization (CWE) were recorded on days 3, 7, 14 and 21. Pain level, number of analgesics used and bleeding status were recorded for the first 7 days. PSTT was measured at baseline and after 1 and 6 months. Colour match scores of the test group were significantly higher than those of the control group at 7 and 14 days. CWE was observed at a higher frequency in the test group than in the control group on day 14. Post-operative bleeding prevalence was lower in the test group than in the control group for the first 2 days. A time-dependent decrease in PSTT was observed at 1 and 6 months in the control group compared with baseline (baseline, 4.23 ± 0.62 mm; 1 month, 4.01 ± 0.68 mm; and 6 months, 3.93 ± 0.69 mm). However, no significant difference was found in the test group (baseline, 4.29 ± 0.64 mm; 1 month, 4.61 ± 0.51 mm; and 6 months, 4.51 ± 0.58 mm). The T-PRF membrane exhibited positive effects on PMWH. T-PRF, which is a promising autogenous matrix for histoconduction, may also be preferred as an autogenous alternative to connective tissue grafts in the treatment of gingival recessions and peri-implant mucosal recessions.

  10. Transfusion of pooled buffy coat platelet components prepared with photochemical pathogen inactivation treatment: the euroSPRITE trial

    NARCIS (Netherlands)

    D.J. van Rhenen (Dirk Jan); S. Marblie (Stephane); M. Laforet (Michel); K. Davis (Kathryn); M. Conlan (Maureen); B. Lioure (Bruno); H. Gulliksson (Hans); J.P. Cazenave; P. Metzel (Peyton); D. Pamphilon (Derwood); L. Corash (Laurence); J. Flament (Jocelyne); P. Ljungman (Per); H. Kluter; H. Vermeij (Hans); V. Mayaudon (Veronique); L. Lin (Lily); M.C. Kappers-Klunne (Mies); D. Buchholz (Don); G.E. de Greef (Georgine)

    2003-01-01

    textabstractA nucleic acid-targeted photochemical treatment (PCT) using amotosalen HCl (S-59) and ultraviolet A (UVA) light was developed to inactivate viruses, bacteria, protozoa, and leukocytes in platelet components. We conducted a controlled, randomized, double-blinded trial in thrombocytopenic

  11. Platelet mimicry

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Hunter, Alan Christy; Peer, Dan

    2016-01-01

    Here we critically examine whether coating of nanoparticles with platelet membranes can truly disguise them against recognition by elements of the innate immune system. We further assess whether the "cloaking technology" can sufficiently equip nanoparticles with platelet-mimicking functionalities...

  12. Compaction agent clarification of microbial lysates

    Science.gov (United States)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  13. Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

    Science.gov (United States)

    Lynch, D M; Howe, S E

    1985-11-01

    Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

  14. In vitro function of random donor platelets stored for 7 days in composol platelet additive solution

    Directory of Open Access Journals (Sweden)

    Gupta Ashish

    2011-01-01

    Full Text Available Background and Aim: Platelets are routinely isolated from whole blood and stored in plasma for 5 days. The present study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C. Materials and Methods: The study sample included 30 blood donors of both sex in State Blood Bank, CSM Medical University, Lucknow. Random donor platelets were prepared by platelet rich plasma method. Whole blood (350 ml was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators, with and without additive solution. Results: Platelet swirling was present in all the units at 22°C on day 7, with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution, whereas platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001 on day 7 without additive solution at 22°C. Conclusion: Our study infers that platelet viability and aggregation were best maintained within normal levels on day 7 of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

  15. Platelets stimulate fibroblast-mediated contraction of collagen gels

    Directory of Open Access Journals (Sweden)

    Lundahl Joachim

    2003-10-01

    Full Text Available Abstract Background Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF and transforming growth factor-β (TGF-β contribute to this effect. Methods Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing, were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β1 concentrations were measured in culture supernatants by ELISA. Results Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM of initial area vs. 48.0% ± 0.4 at 48 hours; P 1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction. Conclusion We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.

  16. Storage of platelets: effects associated with high platelet content in platelet storage containers.

    Science.gov (United States)

    Gulliksson, Hans; Sandgren, Per; Sjödin, Agneta; Hultenby, Kjell

    2012-04-01

    A major problem associated with platelet storage containers is that some platelet units show a dramatic fall in pH, especially above certain platelet contents. The aim of this study was a detailed investigation of the different in vitro effects occurring when the maximum storage capacity of a platelet container is exceeded as compared to normal storage. Buffy coats were combined in large-volume containers to create primary pools to be split into two equal aliquots for the preparation of platelets (450-520×10(9) platelets/unit) in SSP+ for 7-day storage in two containers (test and reference) with different platelet storage capacity (n=8). Exceeding the maximum storage capacity of the test platelet storage container resulted in immediate negative effects on platelet metabolism and energy supply, but also delayed effects on platelet function, activation and disintegration. Our study gives a very clear indication of the effects in different phases associated with exceeding the maximum storage capacity of platelet containers but throw little additional light on the mechanism initiating those negative effects. The problem appears to be complex and further studies in different media using different storage containers will be needed to understand the mechanisms involved.

  17. Actualización bibliográfica sobre el uso de preparaciones ricas en plaquetas en la cicatrización de heridas Updated review of the use of platelet rich preparations in wound healing

    Directory of Open Access Journals (Sweden)

    D.A. Mateo de Acosta Andino

    2010-09-01

    Full Text Available Las plaquetas poseen más de 20 factores de crecimiento en sus gránulos. Su secreción promueve la reparación de heridas. La cicatrización es un intrincado proceso en el cual intervienen no solo los factores plaquetarios proangiogénicos sino también factores antiangiogénicos. La preservación por congelación de las plaquetas empleada para su preservación, desestabiliza y activa la membrana plaquetaria alterando sus propiedades. En el campo del tratamiento de heridas recalcitrantes en pacientes debilitados o con enfermedades crónicas, el uso de preparaciones plaquetarias en forma de plasma fresco congelado, las preparaciones plaquetarias sonicadas, las preparaciones congeladas y deshidratadas y el uso de fragmentos de dermis alogénica, confiere efectos beneficiosos, logrando mejores tiempos de cierre en las heridas y menor alteración de la morfología de la cicatriz final. En el presente artículo, describiremos los estudios que han demostrado estos efectos, así como las encrucijadas actuales y los estudios que debemos esperar en un futuro no lejano.Platelets store more than 20 growth factors in their granules. Their secretion promotes wound healing and repair. The intricate wound healing process involves not only the angiogenic platelet factors but also de anti-angiogenic ones. The process of platelet preservation, such as freezing, disrupt the platelet membrane structure and their angiogenic properties causing platelet activation. In the field of wound healing and the management of recalcitrant wounds in debilitated and chronically ill patients, there have been several studies. These studies have focused on the use of the different platelet preparations such as fresh frozen plasma, frozen-dried platelet rich plasma, sonicated platelet rich plasma and the use of alogenic acelular dermis. Platelet preparations posses positive effects on wound healing with little alterations of the final scar tissue. In this article we will describe

  18. Platelet-rich fibrin: Evolution of a second-generation platelet concentrate

    Directory of Open Access Journals (Sweden)

    Sunitha Raja V

    2008-01-01

    Full Text Available Platelet-rich plasma (PRP is a platelet concentrate that has been used widely to accelerate soft-tissue and hard-tissue healing. The preparation of PRP has been described by several authors. Platelet-rich fibrin (PRF was first described by Choukroun et al. in France. It has been referred to as a second-generation platelet concentrate, which has been shown to have several advantages over traditionally prepared PRP. Its chief advantages include ease of preparation and lack of biochemical handling of blood, which makes this preparation strictly autologous. This article describes the evolution of this novel platelet concentrate, referred to as PRF.

  19. Platelet activation in pregnancy-induced hypertension.

    Science.gov (United States)

    Karalis, Ioannis; Nadar, Sunil K; Al Yemeni, Eman; Blann, Andrew D; Lip, Gregory Y H

    2005-01-01

    Although excess platelet activation, as indicated by increased plasma beta thromboglobulin (beta-TG), has been shown in pregnancy-induced hypertension (PIH), platelet adhesion, platelet morphology and a comparison of platelet and soluble (plasma) levels of the adhesion molecules P-selectin (pPsel and sPsel, respectively) have not been studied. We conducted a cross-sectional study of 35 consecutive women with PIH (age 31+/-6 years), 31 consecutive women with normotensive pregnancies (age 29+/-5 years) and 30 normotensive non pregnant women (age 30+/-5 years). Platelet adhesion was studied in vitro by binding to fibrinogen-coated microwells, platelet morphology [mass and volume by flow cytometry], whole-platelet P-selectin (pPsel) by ELISA of the lysate of 2 x 10(8) cells, and the plasma markers soluble P-selectin (sP-sel) and beta-TG, by ELISA. The women with PIH had significantly raised sPsel, pPsel and (as expected) beta-TG (all p<0.05), when compared to the normotensive pregnant women and controls. However, in PIH platelet adhesion was similar to that in the normotensive pregnancy, but still higher than the normal controls (p<0.001). There was no difference among the three groups with respect to platelet mass and volume. pPsel and platelet adhesion correlated with gestational age and with systolic and diastolic blood pressure (all p<0.05). Increased platelet activation and adhesion develop during normal pregnancy, with some indices being further altered in PIH.

  20. Distribution of In-111 in granulocyte and other cellular elements of blood (CEB) in human In-111-labeled mixed white cell (MWC) and platelet preparations

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Chowdhury, S.; Brown, M.L.; Wahner, H.W.

    1984-01-01

    A large number of platelets (PLT), red blood cells (RBC) are present along with granulocyte (GC) in In-111 in CEB was determined by Ficoll-Hypaque gradient (FHG) centrifugation of In-111-MWC and PLT preparation as a quality control procedure. MWC were separated by sedimentation with hydroxyethyl starch; PLT by differential centrifugation. MWC and PLT were labeled with In-111-oxine in saline, ACD-saline or with In-111-tropolone in 0.5 ml of ACD-plasma. 0.3-0.5 ml of labeled cell suspended in plasma was layered on 3 ml FHG of two densities (1.119 and 1.077 gm/ml) and spun in a clear polystyrene tube at 1800 G for 30 min. Four layers (plasma, PLT, GC, and RBC) were separated, and In-111 radioactivity in each fraction was determined with a gamma counter. Simultaneously cell types in MWC and PLT preparations were determined by Coulter counter and differential counting. Most of In-111 in In-MWC is associated with the PLT and RBC, GC/lymphocyte ratio is 6/4. GC has higher extraction efficiency than RBC and PLT. PLT preparation is pure and (96 +- 3)% of In-111 is bound to PLT, (4 +- 3)% to RBC and (0.2 +- 0.1)% to GC; PLT preparation contains PLT (97 +- 3)%, RBC (4 +- 3)% and GC (0.2 +- 0.1)%

  1. The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation.

    Science.gov (United States)

    Kawase, Tomoyuki; Kamiya, Mana; Kobayashi, Mito; Tanaka, Takaaki; Okuda, Kazuhiro; Wolff, Larry F; Yoshie, Hiromasa

    2015-05-01

    Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37°C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment. © 2014 Wiley Periodicals, Inc.

  2. Platelet count

    Science.gov (United States)

    The normal number of platelets in the blood is 150,000 to 400,000 platelets per microliter (mcL) or 150 to 400 × 10 9 /L. Normal value ranges may vary slightly. Some lab use different measurements or ...

  3. Responsiveness of platelets during storage studied with flow cytometry - formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion

    OpenAIRE

    Södergren, Anna; Tynngård, Nahreen; Berlin, Gösta; Ramström, Sofia

    2016-01-01

    Background and ObjectivesStorage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Materials and MethodsActivation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lyso...

  4. PDGF-AB rich-trombocyte lysate supplementation from breast cancer patients increased the proliferation of breast cancer stem cells

    Directory of Open Access Journals (Sweden)

    Wiwi A. Kartolo

    2018-05-01

    Full Text Available Background: Thrombocytosis in breast cancer (BC patient was thought to play a role in the invasiveness of breast cancer stem cells (BCSCs. Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. This study was aimed to analyze the effect of platelet lysate (PL as well as its PDGF-AB content as a tumor microenvironment on (CD24-/CD44+ BCSC proliferation.Methods: This was an experimental study that treated culture of BCSCs with PL from breast cancer (BC patients or healthy donors. Venous blood from all subjects were subjected to prior hematology test and then processed to obtain platelet rich plasma  (PRP. Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF-AB contents in PL were measured. PL at concentrations of 0.01% (v/v was supplemented into DMEM-F12 medium and used for culturing BCSCs (CD24-/CD44+ cells. After 48 hours, total cell count, population doubling time (PDT, and cell viability were calculated and their correlation with platelet count and PDGF-AB levels were analyzed.Results: BC patients (n=5 had higher platelet counts and PDGF-AB levels in PL compared to healthy donors (n=15, (p=0.02. PL from BC patients could stimulate the proliferation of BCSCs higher than healthy donors (p<0.001 and showed lower PDT value (p=0.001. Cell proliferation and PDT showed strong correlation with PDGF-AB level. This observation suggests that PDGF-AB has a role on BCSCs proliferation. PL showed no effect on BCSCs viability.Conclusion: Breast cancer patient platelet lysate stimulated BCSC proliferation.

  5. Platelet Donation

    Science.gov (United States)

    ... During your donation you can relax, watch a movie, listen to music…in a few hours you’ ... requirements may become eligible to donate platelets. Please review our eligibility requirements as some states require parental ...

  6. Platelet-rich fibrin matrix for facial plastic surgery.

    Science.gov (United States)

    Sclafani, Anthony P; Saman, Masoud

    2012-05-01

    Platelets are known primarily for their role in hemostasis, but there is increasing interest in the effect of platelets on wound healing. Platelet isolates such as platelet-rich plasma have been advocated to enhance and accelerate wound healing. This article describes the use of a novel preparation, platelet-rich fibrin matrix (PRFM), for facial plastic surgery applications such as volume augmentation, fat transfer supplementation, and as an adjunct to open surgical procedures. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Heterogeneity of rabbit platelets

    International Nuclear Information System (INIS)

    Karpatkin, S.

    1978-01-01

    Rabbits were injected intravenously with a cohort platelet label, 75 Se-selenomethionine. Platelet-rich plasma was separated into five different platelet density fractions on each of seven days by repetitively centrifuging the same sample of platelet-rich plasma at increasing gravitational force. The heaviest platelet sediment fraction was enriched with larger platelets. The lightest platelet sediment fraction was enriched with smaller platelets. Incorporation of isotope into the heaviest platelet fraction was considerably greater than incorporation into the lightest platelet fraction. The mean platelet survival of the lightest two fractions was significantly shorter than that of the heaviest three fractions. SDS-polyacrylamide gel electrophoresis of the platelet cell sap generally revealed 10 prominent protein bands for the heaviest platelet fractions. The lightest platelet fraction had six absent to markedly diminished platelet proteins. The data are compatible with two models, (1) heavy-large platelets are, on average, young platelets which become lighter-smaller platelets while losing platelet membranes and cell sap components with time. (2) Heavy-large platelets and light-small platelets are produced independently by specific megakarocytes. The heavy-large platelets incorporate more isotope that lighter-smaller platelets (possibly because of their megakarocyte precursor). However, they are released earlier into the circulation than lighter-smaller platelets and are therefore younger platelets. The light-smaller platelets which are released later into the circulation have a shorter survival. (author)

  8. Imipramine binding in subpopulations of normal human blood platelets

    International Nuclear Information System (INIS)

    Arora, R.C.; Meltzer, H.Y.

    1984-01-01

    Imipramine binding was studied in platelet membranes isolated with different proportions of heavy (young) and light (old) platelets. The B/sub max/, a measure of the number of binding sites, was greater in the heavier platelets than in the light platelets. However, the dissociation constant K/sub d/ (a reflection of the affinity of imipramine binding) was greater in the lighter platelets compared to the heavy platelets. These results indicate that differences in K/sub d/ and B/sub max/ in particular membrane preparation, could be due to the differences in the relative proportion of heavy and light platelets

  9. In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release.

    Science.gov (United States)

    Huber, Stephany Cares; Cunha Júnior, José Luiz Rosenberis; Montalvão, Silmara; da Silva, Letícia Queiroz; Paffaro, Aline Urban; da Silva, Francesca Aparecida Ramos; Rodrigues, Bruno Lima; Lana, José Fabio Santos Duarte; Annichino-Bizzacchi, Joyce Maria

    2016-01-01

    The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.

  10. Platelet Function Tests

    Science.gov (United States)

    ... Patient Resources For Health Professionals Subscribe Search Platelet Function Tests Send Us Your Feedback Choose Topic At ... Also Known As Platelet Aggregation Studies PFT Platelet Function Assay PFA Formal Name Platelet Function Tests This ...

  11. Platelet antibodies blood test

    Science.gov (United States)

    This blood test shows if you have antibodies against platelets in your blood. Platelets are a part of the blood ... Chernecky CC, Berger BJ. Platelet antibody - blood. In: Chernecky ... caused by platelet destruction, hypersplenism, or hemodilution. ...

  12. In vitro function of random donor platelets stored for 7 days in composol platelet additive solution

    Directory of Open Access Journals (Sweden)

    Gupta Ashish

    2011-01-01

    Full Text Available Background and Aim: Platelets are routinely isolated from whole blood and stored in plasma for 5 days. This study was done to assess the in vitro function of random donor platelets stored for 7 days in composol platelet additive solution at 22°C. Materials and Methods: The study sample included 30 blood donors of both sex in State Blood Bank, C S M Medical University, Lucknow. Random donor platelets were prepared by the platelet-rich plasma method. Whole blood (350 ml was collected in anticoagulant Citrate Phosphate Dextrose Adenine triple blood bags. Random donor platelets were stored for 7 days at 22°C in platelet incubators and agitators with and without additive solution. Results: Platelet swirling was present in all the units at 22°C on day 7 with no evidence of bacterial contamination. Comparison of the mean values of platelet count, platelet factor 3, lactate dehydrogenase, pH, glucose and platelet aggregation showed no significant difference in additive solution while platelet factor 3, glucose and platelet aggregation showed significant difference (P < 0.001 on day 7 without additive solution at 22°C. Conclusion: Our study infers that the platelet viability and aggregation were the best maintained within normal levels on day 7 of storage in platelet additive solution at 22°C. Thus, we may conclude that in vitro storage of random donor platelets with an extended shelf life of 7 days using platelet additive solution may be advocated to improve the inventory of platelets.

  13. Platelet modulation of human neutrophil functions

    Energy Technology Data Exchange (ETDEWEB)

    McGarrity, S.T.; Hyers, T.M.; Webster, R.O.

    1986-03-01

    The combined presence of platelets (PLTS) and neutrophils (PMN) at inflammatory sites has led to examination of the hypothesis that interaction of these cells modulates their responses to stimuli. Gel-filtered human PLTS (GFP) were found to inhibit N-formyl-met-leu-phe (FMLP) and phorbol myristate acetate (PMA) stimulated PMN O/sub 2//sup -/ generation in a concentration-dependent fashion. The heat-stable inhibitory activity was present in the supernatants of GFP after incubation with FMLP (10/sup -7/M), thrombin (0.5 U/ml) or ADP (20 ..mu..M), suggesting a role for PLT release products. PLT lysates added to PMN produced up to 80% inhibition of O/sub 2//sup -/ generation for PMA and 40% for FMLP. Like GFP, lysates failed to scavenge O/sub 2/..pi.. produced by xanthine oxidase-hypoxanthine. The inhibitory activity could not be ascribed to serotonin or adenosine. PLT lysates failed to compete with /sup 3/H-FMLP for binding to PMN. Sephadex G-200 fractionation of PLT lysates releaved two peaks of inhibitory activity with apparent Mr > 200,000 and < 14,000 Daltons. Pretreatment of PMN with PLT lysates also results in a concentration-dependent inhibition of degranulation provoked by FMLP (2 x 10/sup -7/M) or PMA (2 ng/ml) and PMN chemotaxis to FMLP (10/sup -8/M). These studies indicate that preformed PLT mediator(s) released in response to physiological stimuli may limit tissue damage by PMN at sites of inflammation.

  14. Preparation of a new composite combining strengthened β-tricalcium phosphate with platelet-rich plasma as a potential scaffold for the repair of bone defects

    Science.gov (United States)

    WANG, CHENGGONG; ZHONG, DA; ZHOU, XING; YIN, KE; LIAO, QIANDE; KONG, LINGYU; LIU, ANSONG

    2014-01-01

    β-tricalcium phosphate (β-TCP) and platelet-rich plasma (PRP) are commonly used in bone tissue engineering. In the present study, a new composite combining strengthened β-TCP and PRP was prepared and its morphological and mechanical properties were investigated by scanning electron microscopy (SEM) and material testing. The biocompatibility was evaluated by measuring the adhesion rate and cytotoxicity of bone marrow stromal cells (BMSCs). The strengthened β-TCP/PRP composite had an appearance like the fungus Boletus kermesinus with the PRP gel distributed on the surface of the micropores. The maximum load and load intensity were 945.6±86.4 N and 13.1±0.5 MPa, which were significantly higher than those of β-TCP (110.1±14.3 N and 1.6±0.2 MPa; P96% after 24 h, with a cell cytotoxicity value of zero. SEM micrographs revealed that following seeding of BMSCs onto the composite in high-glucose Dulbecco’s modified Eagle’s medium culture for two weeks, the cells grew well and exhibited fusiform, spherical and polygonal morphologies, as well as pseudopodial connections. The strengthened β-TCP/PRP composite has the potential to be used as a scaffold in bone tissue engineering due to its effective biocompatibility and mechanical properties. PMID:25187800

  15. Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

    OpenAIRE

    Bayer, Andreas; Tohidnezhad, Mersedeh; Lammel, Justus; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

    2017-01-01

    Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF?) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiatio...

  16. Platelet Donation

    Science.gov (United States)

    ... time’ to unwind from the daily stresses of life while helping save lives. What are the benefits to donating platelets? Knowing you’re helping cancer ... of your arm. That pinch is similar to what you will feel when the needle is ... compared to a traditional whole blood donation so some donors find it to ...

  17. Some aspects of rat platelet and serum phospholipase A2 activities

    NARCIS (Netherlands)

    Aarsman, A.J.; Roosenboom, C.F.P.; Geffen, G.E.W. van; Bosch, H. van den

    1985-01-01

    Rat platelet lysate contained appreciable phospholipase A2 activity. In agreement with literature data this enzymatic activity eluted in the void volume of a Sephadex G-100 column. When the void volume peak was chromatographed over a Matrex gel blue A column, part of the phospholipase A2 activity

  18. EXTENDED STORAGE OF BUFFY-COAT PLATELET CONCENTRATES IN PLASMA OR A PLATELET ADDITIVE SOLUTION

    Science.gov (United States)

    Slichter, Sherrill J.; Bolgiano, Doug; Corson, Jill; Jones, Mary Kay; Christoffel, Todd; Bailey, S. Lawrence; Pellham, Esther

    2014-01-01

    Background Platelet concentrates prepared from whole blood in the U.S. are made using the platelet-rich-plasma (PRP) method. The platelet concentrates must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, platelet concentrates are made using the buffy-coat (BC) method from whole blood held overnight at 22°C and storage times may be up to 7 days. Our studies were designed to determine how long BC platelets can be stored in plasma or Plasmalyte while meeting the FDA’s post-storage viability criteria. Study Design, Materials, And Methods Normal subjects donated whole blood that was stored at 22°C for 22 ± 2 hours prior to preparation of BC platelets. Platelets were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65% or 80%. Radiolabeled autologous stored versus fresh platelet recoveries and survivals were assessed as well as post-storage in vitro assays. Results BC platelets stored in either plasma or 65% Plasmalyte met FDA post-storage platelet recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored platelet recoveries and survivals correlated with the same donor’s fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted post-storage platelet recoveries, while annexin V binding best predicted platelet survivals. Conclusion BC platelets stored in either plasma or 65% Plasmalyte meet FDA’s post-storage viability criteria for 6 days. PMID:24673482

  19. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    Science.gov (United States)

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Congenital platelet function defects

    Science.gov (United States)

    ... pool disorder; Glanzmann's thrombasthenia; Bernard-Soulier syndrome; Platelet function defects - congenital ... Congenital platelet function defects are bleeding disorders that cause reduced platelet function. Most of the time, people with these disorders have ...

  1. Comparative evaluation of platelet count and antimicrobial efficacy of injectable platelet-rich fibrin with other platelet concentrates: An in vitro study

    Directory of Open Access Journals (Sweden)

    Prerna Ashok Karde

    2017-01-01

    Full Text Available Background: Platelet concentrates are used in various medical procedures to promote soft- and hard-tissue regeneration. In recent times, their antimicrobial efficacy is also explored. However, various platelet concentrates have evolved which differ in the centrifugation protocols. One such recently introduced platelet concentrate is injectable platelet-rich fibrin (i-PRF concentrate. Hence, the aim was to evaluate the antimicrobial property, and platelet count of i-PRF in comparison to other platelet concentrates, i.e., PRF, platelet-rich plasma (PRP, and control (whole blood. Materials and Methods: Blood samples were obtained from 10 chronic generalized marginal gingivitis patients. Platelet concentrates were prepared using standardized centrifugation protocol. Platelet count was evaluated by manual counting method using smear preparation of each sample. Subsequently, antimicrobial activity against oral bacteria was examined on blood agar using disc diffusion method to quantify the inhibitory effects. Results: Statistical significance was analyzed by one-way analysis of variance (ANOVA. P 0.05. i-PRF showed statistically significant difference (P < 0.001 in platelet count when compared to control. It was also significant when compared to PRP (P < 0.01, PRF (P < 0.001. Conclusion: i-PRF has maximum antimicrobial efficacy and higher platelet count in comparison to other platelet concentrates, thereby indicating to have a better regenerative potential then others.

  2. The effect of centrifugation speed and time on pre-analytical platelet activation.

    Science.gov (United States)

    Söderström, Anna C; Nybo, Mads; Nielsen, Christian; Vinholt, Pernille J

    2016-12-01

    The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation. Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean platelet volume (MPV), immature platelet fraction (IPF), and platelet distribution width (PDW) were measured. Platelet aggregation in platelet-rich plasma induced by arachidonic acid (AA), ADP or thrombin receptor activator peptide-6 (TRAP) was tested by 96-well aggregometry. The median percentage of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%-53%] and 56% (IQR, 31%-78%), respectively (p=0.82). Platelet-rich plasma prepared at 100-250 g for 10 min had significantly lower platelet P-selectin expression (11%-15%), pcentrifuged. In platelet-rich plasma, increasing centrifugation speed significantly increased platelet yield but decreased contamination from other blood cells, platelet composition was altered as platelet parameters (MPV, IPF, and PDW) was lowered. Platelet aggregation was not affected by the centrifugation speed platelet-rich plasma was prepared. Proportional to centrifugation speed, platelets in plasma and platelet-rich plasma were activated with centrifugation speed, cell content and composition changed while platelet aggregation was unaltered.

  3. The kinetics of short-lived Indium-111 radiolabelled platelets

    International Nuclear Information System (INIS)

    Peters, A.M.; Saverymuttu, S.H.; Bell, R.N.; Lavender, J.P.

    1985-01-01

    We have studied the kinetics of autologous 111 In-labelled platelets in patients with reduced platelet life span ( 111 In-labelled platelets in patients with severe thrombocytopenia. Intrasplenic platelet transit time (t) was calculated by compartmental and deconvolution analysis. In patients with a mean platelet life span of less than a few h, compartmental analysis may not be valid and so only deconvolution analysis was applied. There was a close correlation between values of t given by the two approaches (r=0.88, n=18, P<0.001). In some patients with severely reduced mean platelet life span (MPLS), the deconvolved splenic platelet clearance curves appeared to approach an asymptote, the relative magnitude of which was indicative of the irreversible extraction fraction by the spleen of incoming platelets. In othe patients with severely reduced MPLS resulting from abnormal intra-hepatic platelet destruction, the deconvolved splenic curves resembled the normal. The intrasplenic platelet transit time showed no clear relationship with other parameters. It was concluded that platelet pooling within the spleen is normal in patients with reduced platelet life span,including idiopathic thrombocytopenic purpura, even when the predominant site of destruction is the spleen, and that platelets are not delayed in transit through the spleen in preparation of their removal from the circulation and ultimate destruction. (author)

  4. Kinetics of short-lived Indium-111 radiolabelled platelets

    Energy Technology Data Exchange (ETDEWEB)

    Peters, A.M.; Saverymuttu, S.H.; Bell, R.N.; Lavender, J.P. (Hammersmith Hospital, London, U.K.)

    1985-01-01

    We have studied the kinetics of autologous /sup 111/In-labelled platelets in patients with reduced platelet life span (<4.5 d), most of whom were thrombocytopenic, and of homologous /sup 111/In-labelled platelets in patients with severe thrombocytopenia. Intrasplenic platelet transit time (t) was calculated by compartmental and deconvolution analysis. In patients with a mean platelet life span of less than a few h, compartmental analysis may not be valid and so only deconvolution analysis was applied. There was a close correlation between values of t given by the two approaches (r=0.88, n=18, P<0.001). In some patients with severely reduced mean platelet life span (MPLS), the deconvolved splenic platelet clearance curves appeared to approach an asymptote, the relative magnitude of which was indicative of the irreversible extraction fraction by the spleen of incoming platelets. In other patients with severely reduced MPLS resulting from abnormal intra-hepatic platelet destruction, the deconvolved splenic curves resembled the normal. The intrasplenic platelet transit time showed no clear relationship with other parameters. It was concluded that platelet pooling within the spleen is normal in patients with reduced platelet life span,including idiopathic thrombocytopenic purpura, even when the predominant site of destruction is the spleen, and that platelets are not delayed in transit through the spleen in preparation of their removal from the circulation and ultimate destruction.

  5. Platelet fibrinogen binding in Basset Hound Hereditary Thrombopathy

    International Nuclear Information System (INIS)

    Patterson, W.; Estry, D.; Schwartz, K.; Bell, T.

    1986-01-01

    Platelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) display a thrombasthenia-like aggregation defect but have been shown to have normal amounts of platelet membrane glycoproteins IIb and IIIa (GP IIb-IIIa). In order to investigate the possibility of a functionally abnormal GPIIb-IIIa complex, which might be unable to bind fibrinogen after stimulation, fibrinogen binding in BHT was evaluated. Two canine fibrinogen preparations were used, one from BHT dogs and one from normal control dogs, as well as a human fibrinogen preparation. Platelets from BHT and normal dogs were activated with 1 x 10 -5 M ADP in the presence of 125 I-labeled fibrinogen and the surface bound radioactivity quantitated. For all fibrinogen preparations, the amount of fibrinogen bound by BHT platelets was not significantly different than that bound by normal dog platelets. BHT platelets bound 23,972 +/- 3612 and normal dog platelets bound 23,033 +/- 3971 molecules of fibrinogen per platelet. The BHT platelet aggregation defect does not seem to be caused by a functionally abnormal GP IIb-IIIa complex, since BHT platelets bind normal amounts of fibrinogen. The results suggest that fibrinogen binding is not sufficient for platelet aggregation, and other factors, perhaps receptor mobility and membrane phospholipid content should be investigated in BHT

  6. Responsiveness of platelets during storage studied with flow cytometry--formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion.

    Science.gov (United States)

    Södergren, A L; Tynngård, N; Berlin, G; Ramström, S

    2016-02-01

    Storage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Activation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lysosome-associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) exposure were assessed by flow cytometry on platelets in different subpopulations in resting state or following stimulation with platelet agonists (cross-linked collagen-related peptide (CRP-XL), PAR1- and PAR4-activating peptides). The ability to form subpopulations upon activation was significantly decreased already at day 5 for some agonist combinations. The agonist-induced exposure of PS and LAMP-1 also gradually decreased with time. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. In addition, agonist-induced LAMP-1 expression clearly discriminated platelet concentrates with reduced swirling from those with retained swirling. This suggests that LAMP-1 could be a good marker to capture changes in activation capacity in stored platelets. The platelet activation potential seen as LAMP-1 exposure and fragmentation into platelet subpopulations is potential sensitive markers for the platelet storage lesion. © 2015 International Society of Blood Transfusion.

  7. In vitro effect of sodium nitrite on platelet aggregation in human platelet rich plasma--preliminary report.

    Science.gov (United States)

    Kadan, M; Doğanci, S; Yildirim, V; Özgür, G; Erol, G; Karabacak, K; Avcu, F

    2015-10-01

    The role of nitrates and nitric oxide on platelet functions has obtained an increasing attention with respect to their potential effects on cardiovascular disorders. In this study we aimed to analyze the effect of sodium nitrite on platelet functions in human platelets. This in vitro study was designed to show the effect of sodium nitrite on platelet functions in seven healthy volunteers. Blood samples were centrifuged to prepare platelet rich plasma and platelet poor plasma. Platelet rich plasma was diluted with the platelet poor plasma to have a final count of 300,000 ± 25,000 platelets. Platelet rich plasma was incubated with six different increasing doses (from 10 μM to 5 mM) of sodium nitrite for 1 hour at 37°C. Then stimulating agents including collagen (3 μg ml-1), adenosine diphosphate (10 μM), and epinephrine (10 μM) were added to the cuvette. Changes in light transmission were observed for 10 minutes. In addition spontaneous aggregation were performed in control group with all aggregating agents separately. Effect of sodium nitrite on agonist-induced platelet aggregation depends on the concentration of sodium nitrite. Compared with control group, agonist-induced platelet aggregations were significantly suppressed by sodium nitrite at the concentration of 5, 1.0 and 0.5 mM. Our results suggested that sodium nitrite has inhibitory effects in vitro on platelet aggregation in a dose-dependent manner.

  8. Platelet-functionalized three-dimensional polye-epsilon-caprolactone fibrous scaffold prepared using centrifugal spinning for delivery of growth factors

    Czech Academy of Sciences Publication Activity Database

    Rampichová, Michala; Buzgo, M.; Míčková, Andrea; Vocetková, Karolína; Sovková, Věra; Lukášová, Věra; Filová, Eva; Rustichelli, Franco; Amler, Evžen

    2017-01-01

    Roč. 12, č. 2017 (2017), s. 347-361 E-ISSN 1178-2013 R&D Projects: GA ČR(CZ) GA15-15697S; GA MŠk(CZ) LO1508 Institutional support: RVO:68378041 Keywords : centrifugal spinning * 3D scaffold * platelets Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Nano-materials (production and properties) Impact factor: 4.300, year: 2016

  9. Radiation-induced volatile hydrocarbon production in platelets

    International Nuclear Information System (INIS)

    Radha, E.; Vaishnav, Y.N.; Kumar, K.S.; Weiss, J.F.

    1989-01-01

    Generation of volatile hydrocarbons (ethane, pentane) as a measure of lipid peroxidation was followed in preparations from platelet-rich plasma irradiated in vitro. The hydrocarbons in the headspace of sealed vials containing irradiated and nonirradiated washed platelets, platelet-rich plasma, or platelet-poor plasma increased with time. The major hydrocarbon, pentane, increased linearly and significantly with increasing log radiation dose, suggesting that reactive oxygen species induced by ionizing radiation result in lipid peroxidation. Measurements of lipid peroxidation products may give an indication of suboptimal quality of stored and/or irradiated platelets

  10. Platelet-rich fibrin or platelet-rich plasma – which one is better? an opinion

    Directory of Open Access Journals (Sweden)

    Shweta Bansal

    2017-01-01

    Full Text Available The healing of hard and soft tissue in mediated by a wide range of intracellular and extracellular events that are regulated by signaling proteins. Platelets can play a crucial role in periodontal regeneration as they are the reservoirs of growth factors and cytokines which are the key factors for regeneration of bone and maturation of soft tissue. Platelet-rich plasma (PRP is first generation platelet concentrate. However, the short duration of cytokine release and its poor mechanical properties have resulted in search of new material. Platelet-rich fibrin (PRF is a natural fibrin-based biomaterial prepared from an anticoagulant-free blood harvest without any artificial biochemical modification (no bovine thrombin is required that allows obtaining fibrin membranes enriched with platelets and growth factors. The slow polymerization during centrifugation, fibrin-based structure, ease of preparation, minimal expense makes PRF somewhat superior in some aspect to PRP.

  11. The use of lumbar epidural injection of platelet lysate for treatment of radicular pain

    NARCIS (Netherlands)

    Centeno, Christopher; Markle, Jason; Dodson, Ehren; Stemper, Ian; Hyzy, Matthew; Williams, Christopher; Freeman, Michael

    2017-01-01

    BACKGROUND: Epidural steroid injections (ESI) are the most common pain management procedure performed in the US, however evidence of efficacy is limited. In addition, there is early evidence that the high dose of corticosteroids used can have systemic side effects. We describe the results of a case

  12. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Effects of Platelets on Platelet Concentrate Product on the Activation of Human Peripheral Blood Monocyte Cells

    Directory of Open Access Journals (Sweden)

    N Sadat Razavi Hoseini

    2016-02-01

    Full Text Available Introduction: Monocytes can interact with platelets due to their surface molecules such as P-selectin glycoprotein ligand-1 (PSGL-1, and form monocyte-platelet complex. In the present study, the effects of platelets interaction of platelet concentrates (PCs and peripheral blood monocytes were investigated in vitro as a model to predict the probable interactions of these cells and consequently activation of monocytes. Methods: In this experimental study, units of whole blood and PCs were prepared from Tehran Blood Transfusion Center. After isolation of monocytes from the whole blood, these cells were treated with PC- derived platelets. The activation of monocytes was assessed before and after treatment by the analysis of the respiratory burst of monocytes using dihydrorhodamine 123 (DHR-123. The study data were analyzed using the non-parametric test of Wilcoxon. Results: The purity of monocytes was determined as 86.1±2 using NycoPrep method. The respiratory burst of monocytes was increased after exposure with platelets. In fact, the difference was significant when platelets were used on the 5th day of storage (P=0.001. Conclusions: The study findings revealed that platelets have an efficient capacity to stimulate and activate monocytes. The possible involvement of molecules in the interaction of platelet-monocyte demand to be further studied in future.

  14. Evaluation of platelet aggregation in platelet concentrates: storage implications

    Directory of Open Access Journals (Sweden)

    Neiva Teresinha J.C.

    2003-01-01

    Full Text Available The use of hemo-derivatives is nowadays a fundamentally important therapeutic modality in the exercise of medicine. Among the various hemo-components employed, we have the platelet concentrate (PC, indicated in cases of hemorrhagic disturbances. We previously showed that platelet function in blood donors is reduced in their screening phase and after the separation process of PCs. Currently, we are providing evidence for the existence of biochemical and functional changes in PC preparations stored for three days at temperatures of 20 ± 2 ºC. Platelet concentrates from 40 healthy donors, collected in CPD anticoagulant and PL-146 polyvinylchloride containers, were examined in order to determine the pH value, pCO2 ,pO2 and lactate concentrations. In addition, the aggregation of platelets with thrombin and collagen were examined to evaluate platelet function. A pH increase from 7.07 ± 0.04 to 7.36 ± 0.07 (p < 0.01 was observed. The pCO2 concentration decreased progressively from 69.2 ± 7.7 mmHg to 28.8 ± 6.2 mmHg (p < 0.001 during the storage period. In contrast, pO2 value increase from 103.4 ± 30.6 to 152.3 ± 24.6 mmHg (p < 0.001 was evidenced during the 48 hours of storage. The lactate concentration increased from 17.97 ± 5.2 to 57.21 ± 5.7 mg/dl (p < 0.001. Platelet aggregation using 0.25 U/ml-thrombin and 2.0 µg/ml-collagen showed significant hypofunction from 61.8 ± 2.7% to 24.8 ± 9.8% and 62.7±5.0 to 33.4± 6.2 (p < 0.001, respectively. We concluded that the evaluated biochemical parameters and the platelet function changed significantly when the platelets were kept under routine storage conditions.

  15. Hypothermia and Platelet Dysfunction

    National Research Council Canada - National Science Library

    Michelson, Alan

    1993-01-01

    ... (the OPlib-Illa complex).3 Circulating platelets are normally in a resting state and, despite the presence of platelet surface GPTh-IX and OPlib-Illa complexes, they bind neither plasma von Willebrand factor nor plasma fibrinogen...

  16. Hypothermia and Platelet Dysfunction

    National Research Council Canada - National Science Library

    Michelson, A

    1994-01-01

    ... (the GPIIb-IIIa complex). Circulating platelets are normally in a resting state and, despite the presence of platelet surface GPIb-IX and GPIIb-IIIa complexes, they bind neither plasma von Willebrand factor nor plasma fibrinogen...

  17. Platelet concentrates for transfusion-metabolic and storage aspects.

    Science.gov (United States)

    Farrugia, A

    1994-01-01

    Transfusion of platelets concentrated from donated blood is an established therapeutic modality in clinical medicine. Over the past 25 years much effort has gone into optimising the conditions for the collection, preparation and storage of platelets for transfusion. Despite significant advances, platelet production is still a costly process requiring a dedicated environment and the use of specially formulated plastic storage containers. A progressive lesion over storage limits the shelf life and the availability of donated platelets, while the need to store platelets in the donor's autologous plasma also results in a loss of valuable fresh plasma for fractionation. Recent studies have addressed the issues of platelet quality and plasma economy by examining the possibility of storing platelets in a synthetic medium. Platelets stored in a variety of crystalloid solutions have been shown to retain in vitro and in vivo properties equivalent or superior to platelets stored in autologous donor plasma. Some additional insight has been gained on the metabolic patterns of stored platelets. In particular, studies have shown that, under these conditions, platelets are unable to oxidise dextrose to any significant extent, and that dextrose is invariably broken down to lactate, irrespective of the oxygen tensions in the platelet's environment. This in turn leads to the metabolic lesion of platelet storage, whereby low pH results in loss of platelet viability. Platelets stored in synthetic dextrose-free media are capable of maintaining aerobic ATP generation, and acetate-a component of many media studied-has been shown to be metabolised by platelets. Similarly, platelets prepared from blood collected into a dextrose-free anticoagulant have satisfactory properties both when suspended in autologous plasma or in a dextrose-free synthetic medium. The requirements for storage in special, high gas-permeable, containers, and for constant agitation during storage, were both found to be

  18. The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors

    International Nuclear Information System (INIS)

    Kalen, Anders; Wahlstroem, Ola; Linder, Cecilia Halling; Magnusson, Per

    2008-01-01

    Platelet derivates and platelet rich plasma have been used to stimulate bone formation and wound healing because of the rich content of potent growth factors. However, not all reports have been conclusive since some have not been able to demonstrate a positive effect. We investigated the interindividual variation of bone morphogenetic proteins (BMPs) in platelets from healthy donors, and the pH-dependent effect on the release of BMPs in preparations of lysed platelets in buffer (LPB). Platelet concentrates from 31 healthy donors were prepared in pH 4.3 and pH 7.4 buffers and investigated with respect to BMP-2, -4, -6, and -7. BMP-2 and BMP-4 were significantly more common in acidic LPBs in comparison with neutral preparations. We also observed a considerable variation among platelet donors with respect to the release of BMPs at pH 4.3 and 7.4. In conclusion, a considerable variation was found among platelet donors, which may be of importance considering the ambiguous results previously reported on osteoblast proliferation and differentiation

  19. Platelet Count and Plateletcrit

    African Journals Online (AJOL)

    strated that neonates with late onset sepsis (bacteremia after 3 days of age) had a dramatic increase in MPV and. PDW18. We hypothesize that as the MPV and PDW increase and platelet count and PCT decrease in sick children, intui- tively, the ratio of MPV to PCT; MPV to Platelet count,. PDW to PCT, PDW to platelet ...

  20. The use of indium-111 platelet scintigraphy in man: comparisons with in vitro tests and in vivo platelet function--a five year experience

    International Nuclear Information System (INIS)

    Ezikowitz, M.D.; Ferri, P.; Pope, C.; Smith, E.O.; Snyder, E.L.

    1985-01-01

    In this paper, the authors present data collected from 540 patients between July 1978 and July 1983. They discuss briefly the method they employed for labeling platelets, emphasizing in vivo and in vitro markers of platelet function used for quality control of platelet preparation. They discuss the application of their technique for identification of mural left ventrical trombi, and review the disparity between in vivo and in vitro tests of platelet function. Then they deal with diagnosis of subacute bacterial endocarditis, deep veonus thrombosis, coronary trombi, left arterial masses, and the use of tomographic imaging. Finally they report changes in platelet function in stored platelets from normal volunteers

  1. Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.

    Science.gov (United States)

    Boswell, Stacie G; Schnabel, Lauren V; Mohammed, Hussni O; Sundman, Emily A; Minas, Tom; Fortier, Lisa A

    2014-01-01

    Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known. Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis. Controlled laboratory study. Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase-3 and -13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1β was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration. Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-β1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1β was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons. Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that

  2. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    Science.gov (United States)

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  3. In vitro model of platelet aggregation in stenotic arteries

    International Nuclear Information System (INIS)

    Morley, D.; Santamore, W.P.

    1988-01-01

    Clinical and experimental evidence suggest a strong relationship between arterial stenosis, platelet aggregation, and subsequent thrombus formation. To facilitate the study of platelet accumulation in stenotic arteries, we developed an in vitro preparation. Arterial segments were perfused with whole citrated blood. A stenosis was created by applying an external plastic constrictor to the artery. Platelet accumulation within the stenosis was assessed by scanning electron microscopy and by radioactive counts from Indium-111 labeled platelets. Utilizing this preparation, 30 carotid arterial segments from 10 mongrel dogs were perfused at 100 mmHg for 15 min. In 10 arteries without a stenosis, scanning electron microscopy and radioactive counts demonstrated little platelet accumulation. In contrast, extensive platelet aggregation was observed in 10 arteries with stenoses. Moreover, in 10 stenotic arteries exposed to the thromboxane mimetic, U46619 (Upjohn Diagnostic Group), scanning electron microscopy and radioactive counts demonstrated a significant increase in platelet deposition. Conversely, we demonstrated a dimunition of platelet accumulation in stenosed arterial segments exposed to the prostacyclin analogue platelet inhibitor, Iloprost. The in vitro preparation allows precise control of hemodynamic variables and makes it possible to perform multiple tests on segments of the same vessel from the same animal

  4. Study on platelet kinetics

    International Nuclear Information System (INIS)

    Yui, Tokuo

    1981-01-01

    Fundamental study: Factors influencing the labeling on human platelets were evaluated and optimal labeling conditions were chosen. Then, platelet survival times were measured and organ distribution of labeled platelets was observed in rat by four different methods. These results were compared with each other. Based on the findings of those studies, the protocol of human platelet labeling with 111 In-oxine for clinical use was established. Clinical study: In normal cases, a platelet survival time and a platelet turnover rate were quite similar to the results from 51 Cr method. In gamma camera imaging, a radioactivity on the heart decreased with the lapse of time, while that on the spleen and liver gradually increased. In patients with idiopathic thrombocytopenic purpura, platelet survival time was markedly shortened and both a platelet turnover rate and an effective production increased. In patient with congestive splenomegaly, a platelet survival time was normal, whereas a platelet pooling on the spleen markedly increased. In patients who were implanted dacron-graft for abdominal aortic aneurysm, a radioactivity accumulated to the graft and a platelet survival time shortened. In a patient with myocardial infarction, the camera imaging clearly showed the thrombus in the left ventricular aneurysm. In three patients with mitral stenosis, thrombi in left atrium were observed at the camera images. Imaging of a platelet distribution and measurement of platelet survival time using 111 In-oxine labeled platelets are considered to be an excellent method for the diagnosis and decision of treatment on various disorders with thrombocytopenia and thrombosis. (J.P.N.)

  5. Vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase

    NARCIS (Netherlands)

    Bleeker, W. K.; Teeling, J. L.; Verhoeven, A. J.; Rigter, G. M.; Agterberg, J.; Tool, A. T.; Koenderman, A. H.; Kuijpers, T. W.; Hack, C. E.

    2000-01-01

    Previously, we observed in a rat model that intravenous administration of intramuscular immunoglobulin preparations induced a long-lasting hypotension, which appeared to be associated with the presence of IgG polymers and dimers in the preparations, but unrelated to complement activation. We found

  6. Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol.

    Science.gov (United States)

    Kay, Jennifer E; Jewett, Michael C

    2015-11-01

    Cell-free metabolic engineering (CFME) is emerging as a powerful approach for the production of target molecules and pathway debugging. Unfortunately, high cofactor costs, limited cofactor and energy regeneration, and low volumetric productivities hamper the widespread use and practical implementation of CFME technology. To address these challenges, we have developed a cell-free system that harnesses ensembles of catalytic proteins prepared from crude lysates, or extracts, of cells to fuel highly active heterologous metabolic conversions. As a model pathway, we selected conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications. Specifically, we engineered a single strain of Escherichia coli to express three pathway enzymes necessary to make meso-2,3-BD (m2,3-BD). We then demonstrated that lysates from this strain, with addition of glucose and catalytic amounts of cofactors NAD+ and ATP, can produce m2,3-BD. Endogenous glycolytic enzymes convert glucose to pyruvate, the starting intermediate for m2,3-BD synthesis. Strikingly, with no strain optimization, we observed a maximal synthesis rate of m2,3-BD of 11.3 ± 0.1 g/L/h with a theoretical yield of 71% (0.36 g m2,3-BD/g glucose) in batch reactions. Titers reached 82 ± 8 g/L m2,3-BD in a 30 h fed-batch reaction. Our results highlight the ability for high-level co-factor regeneration in cell-free lysates. Further, they suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  7. Transcellular lipoxygenase metabolism between monocytes and platelets

    Energy Technology Data Exchange (ETDEWEB)

    Bigby, T.D.; Meslier, N. (Univ. of California, San Francisco (USA))

    1989-09-15

    We have examined the effects of co-culture and in vitro co-stimulation on lipoxygenase metabolism in monocytes and platelets. Monocytes were obtained from the peripheral blood of normal volunteers by discontinuous gradient centrifugation and adherence to tissue culture plastic. Platelets were obtained from the platelet-rich plasma of the same donor. When 10(9) platelets and 2.5 x 10(6) monocytes were co-stimulated with 1 microM A23187, these preparations released greater quantities of 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid, 5(S),12-(S)dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, and leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic (LTC4) when compared with monocytes alone. Release of arachidonic acid, 5-HETE, delta 6-trans-LTB4, and delta 6-trans-12-epi-LTB4 from monocytes was decreased in the presence of platelets. A dose-response curve was constructed and revealed that the above changes became evident when the platelet number exceeded 10(7). Dual radiolabeling experiments with 3H- and 14C-arachidonic acid revealed that monocytes provided arachidonic acid, 5-HETE, and LTA4 for further metabolism by the platelet. Monocytes did not metabolize platelet intermediates detectably. In addition, as much as 1.2 microM 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid and 12(S)-hydroperoxy-10-trans-5,8,14-cis-eicosatetraenoic acid had no effect on monocyte lipoxygenase metabolism. Platelets were capable of converting LTA4 to LTC4, but conversion of LTA4 to LTB4 was not detected. We conclude that the monocyte and platelet lipoxygenase pathways undergo a transcellular lipoxygenase interaction that differs from the interaction of the neutrophil and platelet lipoxygenase pathways. In this interaction monocytes provide intermediate substrates for further metabolic conversion by platelets in an unidirectional manner.

  8. The effect of centrifugation speed and time on pre-analytical platelet activation

    DEFF Research Database (Denmark)

    Söderström, Anna Cecilia; Nybo, Mads; Nielsen, Christian

    2016-01-01

    BACKGROUND: The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation....... METHODS: Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean...... of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%-53%] and 56% (IQR, 31%-78%), respectively (p=0.82). Platelet-rich plasma prepared at 100-250 g for 10 min had significantly lower platelet P-selectin expression (11%-15%), p...

  9. Indium-111 tropolone, a new tracer for platelet labeling

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Rao, S.A.; Rosemark, J.A.; Chowdhury, S.; Didisheim, P.

    1982-01-01

    Platelets have been labeled with a new neutral, lipid-soluble metal complex of indium 111 ( 111 In) and tropolone. Unlike oxine, which is soluble in ethyl alcohol, tropolone is soluble in isotonic saline. Platelet labeling with 111 In tropolone can be performed in both acid-citrate-dextrose (ACD) plasma and ACD saline within two hours. Labeling efficiency has been 80% to 90%. 111 In tropolone in ACD saline and ACD plasma at tropolone concentrations of 5 and 10 micrograms/ml, respectively, and incubation of the platelets with the tracer at room temperature for 20 minutes were optimal conditions for labeling. The authors have developed an ACD-saline kit for convenient preparation of 111 In-labeled platelets. No adverse effect of 111 In tropolone on platelets has been observed in studies of biodistribution, recovery, and survival of platelets in rabbits and dogs

  10. Indium-111 tropolone, a new tracer for platelet labeling

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Rao, S.A.; Rosemark, J.A.; Chowdhury, S.; Didisheim, P.

    1982-01-01

    Platelets have been labeled with a new neutral, lipid-soluble metal complex of indium 111 ( 111 In) and tropolone. Unlike oxine, which is soluble in ethyl alcohol, tropolone is soluble in isotonic saline. Platelet labeling with 111 In tropolone can be performed in both acid-citrate-dextrose (ACD) plasma and ACD saline within two hours. Labeling efficiency has been 80% to 90%. 111 In tropolone in ACD saline and ACD plasma at tropolone concentrations of 5 to 10 μg/ml, respectively, and incubation of the platelets with the tracer at room temperature for 20 minutes were optimal conditions for labeling. The authors have developed an ACD-saline kit for convenient preparation of 111 In-labeled platelets. No adverse effect of 111 In tropolone on platelets has been observed in studies of biodistribution, recovery, and survival of platelets in rabbits and dogs

  11. Feasibility of Using Multilayer Platelets as Toughening Agents

    Directory of Open Access Journals (Sweden)

    Yuan-Liang Chin

    2009-12-01

    Full Text Available It is known that the toughness of brittle ceramics can be improved significantly with the addition of hard platelets. In the present study, platelet-shape multilayer ceramic laminates are utilized as a toughening agent for alumina ceramics. They are prepared by laminating the BaTiO3-based ceramic tapes. Although the elastic modulus of the BaTiO3-based platelets is lower than that of the alumina matrix, and the platelets are also reactive to alumina at elevated temperatures, the weak platelets are found to exhibit the ability to deflect major matrix cracks by forming a large number of microcrack branches within the platelets, thus achieving the desired toughening effect.

  12. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

    International Nuclear Information System (INIS)

    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-01-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating [ 3 H]thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase

  13. Flavanols and Platelet Reactivity

    Directory of Open Access Journals (Sweden)

    Debra A. Pearson

    2005-01-01

    Full Text Available Platelet activity and platelet-endothelial cell interactions are important in the acute development of thrombosis, as well as in the pathogenesis of cardiovascular disease. An increasing number of foods have been reported to have platelet-inhibitory actions, and research with a number of flavanol-rich foods, including, grape juice, cocoa and chocolate, suggests that these foods may provide some protection against thrombosis. In the present report, we review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent primary hemostasis. Consumption of flavanol-rich cocoa inhibited several measures of platelet activity including, epinephrine- and ADP-induced glycoprotein (GP IIb/IIIa and P-Selectin expression, platelet microparticle formation, and epinephrine-collagen and ADP-collagen induced primary hemostasis. The epinephrine-induced inhibitory effects on GP IIb/IIIa and primary hemostasis were similar to, though less robust than those associated with the use of low dose (81 mg aspirin. These data, coupled with information from other studies, support the concept that flavanols present in cocoa and chocolate can modulate platelet function through a multitude of pathways.

  14. Platelet activation and aggregation

    DEFF Research Database (Denmark)

    Jensen, Maria Sander; Larsen, O H; Christiansen, Kirsten

    2013-01-01

    This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated...

  15. Platelet alloimmunization after transfusion

    DEFF Research Database (Denmark)

    Taaning, E; Simonsen, A C; Hjelms, E

    1997-01-01

    BACKGROUND AND OBJECTIVES: The frequency of platelet-specific antibodies after one series of blood transfusions has not been reported, and in multiply transfused patients is controversial. MATERIALS AND METHODS: We studied the frequency of alloimmunization against platelet antigens in 117 patient...

  16. USE OF BACTERIAL LYSATES IN COMPLEX TREATMENT OF RHINOSINUSITIDIS

    Directory of Open Access Journals (Sweden)

    E.A. Vishneva

    2011-01-01

    Full Text Available Rhinosinusitis is a common term that means inflammation of the mucosa of paranasal and nasal cavities. In the last 10 years there was a three-fold increase in rhinosinusitis’ morbidity rates and annual increase of hospital admissions on the average of 1,5–2%. The prevalence of various forms of rhinosinusitidis among adult population is 5 to 15% and 5% among children. Such ubiquitous spreading is one of the leading factors that stipulate socio-economic burden of the disease. According to various studies, rhinosinusitidis dramatically decrease quality of life: these patients show more inferior results in tests upon pain sensitivity and social activity in comparison with patients with coronary heart disease and COPD; in approximately 26% of patients rhinosinusitidis are followed by development or progression of depression. Rhinosinusitis treatment is one of the hottest issues in modern otorhinolaryngology, that demand more financial support with every year. Achievments in immunology have become grounds for immunocorrection as part of complex treatment of such patients. Bacterial lysates are used as preventive medications.Key words: rhinosinusitidis, immunomodulators, bacterial lysates. (Voprosy sovremennoi pediatrii — Current Pediatrics. — 2011; 10 (6: 157–161

  17. Platelet function in dogs

    DEFF Research Database (Denmark)

    Nielsen, Line A.; Zois, Nora Elisabeth; Pedersen, Henrik D.

    2007-01-01

    Background: Clinical studies investigating platelet function in dogs have had conflicting results that may be caused by normal physiologic variation in platelet response to agonists. Objectives: The objective of this study was to investigate platelet function in clinically healthy dogs of 4...... different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. Methods: Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], 12...... applied. However, the importance of these breed differences remains to be investigated. The PFA-100 method with Col + Epi as agonists, and ADP-induced platelet aggregation appear to be sensitive to ASA in dogs....

  18. Dose- and time-related platelet response with apheresis platelet concentrates and pooled platelets

    Directory of Open Access Journals (Sweden)

    Mohammad Mizanur Rahman

    2017-02-01

    Full Text Available This study was carried out to compare the post-transfusion platelet increment between the apheresis platelet concentrate (n=74 and pooled platelets (n=54. Pre- and post-transfusion platelet count of the recipient were carried out by automated hematology analyzer. In apheresis platelet concentrate group, the mean 24 hours post-transfusion platelet increment was 47 x 109/L which was statistically significant (p<0.001. On the other hand, in pooled platelets group, the mean 24 hours post–transfusions platelet count increment was 11.0 x 109/L which was also statistically significant (p<0.001. This study concluded that the transfusion of apheresis platelet concentrate was more useful than the transfusion of pooled platelets in terms of platelet count increment and requirement of donor.

  19. Imaging thrombus with radiolabelled monoclonal antibody to platelets

    International Nuclear Information System (INIS)

    Loutfi, I.; Peters, A.M.; Lavender, J.P.; Epenetos, A.A.

    1988-01-01

    Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111 In labelled monoclonal antibody, P 256 , directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P 256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P 256 was radiolabelled with 111 In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo - that is, by direct intravenous injection of P 256 - in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111 In kinetics recorded after intravenous P 256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P 256 , three had documented thrombus, tow of whom gave positive results on P 256 platelet scintigraphy. The third subject had chromic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis. 3 refs. (Author)

  20. Measurement of platelet aggregation, independently of patient platelet count

    DEFF Research Database (Denmark)

    Vinholt, P J; Frederiksen, H; Hvas, A-M

    2017-01-01

    with collagen-related peptide). Platelet aggregation had a negative predictive value of 100% for a bleeding tendency among patients. Conclusion The established platelet aggregation assay was applicable for thrombocytopenic patients, and improved the identification of bleeding risk.......Essentials •Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. •A flow cytometric platelet aggregation test independent of the patient platelet count was made. •Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer....... •High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. Summary Background Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count...

  1. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    International Nuclear Information System (INIS)

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E.

    1987-01-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of [ 3 H]serotonin, or alter the dose-responsive binding of 125 I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF

  2. Platelet storage lesion in interim platelet unit concentrates: A comparison with buffy-coat and apheresis concentrates.

    Science.gov (United States)

    Singh, Sukhi; Shams Hakimi, Caroline; Jeppsson, Anders; Hesse, Camilla

    2017-12-01

    Platelet storage lesion is characterized by morphological changes and impaired platelet function. The collection method and storage medium may influence the magnitude of the storage lesion. The aim of this study was to compare the newly introduced interim platelet unit (IPU) platelet concentrates (PCs) (additive solution SSP+, 40% residual plasma content) with the more established buffy-coat PCs (SSP, 20% residual plasma content) and apheresis PCs (autologous plasma) in terms of platelet storage lesions. Thirty PCs (n=10 for each type) were assessed by measuring metabolic parameters (lactate, glucose, and pH), platelet activation markers, and in vitro platelet aggregability on days 1, 4, and 7 after donation. The expression of platelet activation markers CD62p (P-selectin), CD63 (LAMP-3), and phosphatidylserine was measured using flow cytometry and in vitro aggregability was measured with multiple electrode aggregometry. Higher platelet activation and lower in vitro aggregability was observed in IPU than in buffy-coat PCs on day 1 after donation. In contrast, metabolic parameters, expression of platelet activation markers, and in vitro aggregability were better maintained in IPU than in buffy-coat PCs at the end of the storage period. Compared to apheresis PCs, IPU PCs had higher expression of activation markers and lower in vitro aggregability throughout storage. In conclusion, the results indicate that there are significant differences in platelet storage lesions between IPU, buffy-coat, and apheresis PCs. The quality of IPU PCs appears to be at least comparable to buffy-coat preparations. Further studies are required to distinguish the effect of the preparation methods from storage conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Taurine and platelet aggregation

    International Nuclear Information System (INIS)

    Nauss-Karol, C.; VanderWende, C.; Gaut, Z.N.

    1986-01-01

    Taurine is a putative neurotransmitter or neuromodulator. The endogenous taurine concentration in human platelets, determined by amino acid analysis, is 15 μM/g. In spite of this high level, taurine is actively accumulated. Uptake is saturable, Na + and temperature dependent, and suppressed by metabolic inhibitors, structural analogues, and several classes of centrally active substances. High, medium and low affinity transport processes have been characterized, and the platelet may represent a model system for taurine transport in the CNS. When platelets were incubated with 14 C-taurine for 30 minutes, then resuspended in fresh medium and reincubated for one hour, essentially all of the taurine was retained within the cells. Taurine, at concentrations ranging from 10-1000 μM, had no effect on platelet aggregation induced by ADP or epinephrine. However, taurine may have a role in platelet aggregation since 35-39% of the taurine taken up by human platelets appears to be secreted during the release reaction induced by low concentrations of either epinephrine or ADP, respectively. This release phenomenon would imply that part of the taurine taken up is stored directly in the dense bodies of the platelet

  4. Estriol-induced fibrinolysis due to the activation of plasminogen to plasmin by nitric oxide synthesis in platelets.

    Science.gov (United States)

    Jana, Pradipta; Maiti, Smarajit; Kahn, Nighat N; Sinha, Asru K

    2015-04-01

    Estriol, an oestrogen, at 0.6 nmol/l was reported to inhibit ADP-induced platelet aggregation through nitric oxide synthesis. As nitric oxide has been reported to cause fibrinolysis due to the activation of plasminogen to plasmin, the role of estriol as a fibrinolytic agent was investigated. Also, the mechanism of estriol-induced nitric oxide synthesis in anucleated platelets was investigated. The estriol-induced lysis of platelet-rich plasma (PRP) clot was determined by photography of the clot lysis and by the assay of fibrin degradation products in the lysate and was obtained by SDS-PAGE. Nitric oxide was determined by methemoglobin method. The platelet membrane protein was isolated from the platelets by using Triton X-100 (0.05% v/v). The binding of estriol to the protein was determined by Scatchard plot by using an ELISA for estriol. Estriol at 0.6 nmol/l was found to lyse the clotted PRP due to fibrinolysis that produced fibrin degradation products in the lysate. The amino acid analysis of the platelet membrane protein, which resembles with nitric oxide synthase (NOS) activity, was activated nearly 10-fold over the control in the presence of estriol and was identified to be a human serum albumin precursor (Mr. 69 kDa) that binds to estriol with Kd1 of 6.0 × 10 mol/l and 39 ± 2 molecules of estriol bound the NOS molecule. The estriol-induced nitric oxide is capable of inducing fibrinolysis of the clotted PRP. The binding of estriol to platelet membrane NOS activated the enzyme in the absence of DNA in the platelet.

  5. Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets

    International Nuclear Information System (INIS)

    Peters, A.M.; Lavender, J.P.

    1983-01-01

    The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of ''abnormal'' platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy

  6. Radioimmunoassay of platelet proteins

    International Nuclear Information System (INIS)

    Pepper, D.S.

    1987-01-01

    The radioimmunoassay of platelet-specific proteins has proven to be an excellent way of monitoring platelet activation in vivo. In contrast to earlier methods such as aggregometry, which has been the major tool used in the evaluation of antiplatelet drugs, the RIAs are capable of working with samples which have been subjected to physiological conditions such as haematocrit, oxygen tension, shear rate and ionized calcium concentration. Also, in contrast to aggregometry, no choice of agonist is necessary. Thus, for the first time it has been possible to monitor the effects of therapeutic intervention with drugs upon the platelet release reaction in vivo. It seems reasonable to equate the release reaction in vivo with activation in vivo, though the stimuli necessarily remain unknown. Nevertheless, the fact that a significant number of the compounds mentioned in Table 3 are indeed capable of reducing platelet activation in vivo and that this effect can be measured objectively is a major step forward in our understanding of platelet pharmacology. Two important goals remain to be achieved, however, the establishment of nonhuman animal models for the evaluation of newer compounds in vivo and longer-term goal of proving in the clinical setting the relevance or otherwise of platelet activation per se to the clinical outcome of a particular disease. In this respect, the availability of accurate, reliable and specific radioimmunoassays has a central role

  7. Concentration of platelets and growth factors in platelet-rich plasma from Goettingen minipigs.

    Science.gov (United States)

    Jungbluth, Pascal; Grassmann, Jan-Peter; Thelen, Simon; Wild, Michael; Sager, Martin; Windolf, Joachim; Hakimi, Mohssen

    2014-01-01

    In minipigs little is known about the concentration of growth factors in plasma, despite their major role in several patho-physiological processes such as healing of fractures. This prompted us to study the concentration of platelets and selected growth factors in plasma and platelet-rich plasma (PRP) preparation of sixteen Goettingen minipigs. Platelet concentrations increased significantly in PRP in comparison to native blood plasma. Generally, significant increase in the concentration of all growth factors tested was observed in the PRP in comparison to the corresponding plasma or serum. Five of the plasma samples examined contained detectable levels of bone morphogenic protein 2 (BMP-2) whereas eleven of the plasma or serum samples contained minimal amounts of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-bb) respectively. On the other hand variable concentrations of bone morphogenic protein 7 (BMP-7) and transforming growth factor β1 (TGF-β1) were measured in all plasma samples. In contrast, all PRP samples contained significantly increased amounts of growth factors. The level of BMP-2, BMP-7, TGF-β1, VEGF and PDGF-bb increased by 17.6, 1.5, 7.1, 7.2 and 103.3 fold, in comparison to the corresponding non-enriched preparations. Moreover significant positive correlations were found between platelet count and the concentrations of BMP-2 (r=0.62, pplatelet-rich plasma of minipigs which might thus serve as a source of autologous growth factors.

  8. Effects of sodium citrate and acid citrate dextrose solutions on cell counts and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel.

    Science.gov (United States)

    Giraldo, Carlos E; Álvarez, María E; Carmona, Jorge U

    2015-03-14

    There is a lack information on the effects of the most commonly used anticoagulants for equine platelet rich plasmas (PRPs) elaboration on cell counts and growth factor release from platelet rich gels (PRGs). The aims of this study were 1) to compare the effects of the anticoagulants sodium citrate (SC), acid citrate dextrose solution A (ACD-A) and ACD-B on platelet (PLT), leukocyte (WBC) and on some parameters associated to platelet activation including mean platelet volume (MPV) and platelet distribution width (PDW) between whole blood, pure PRP (P-PRP) and platelet-poor plasma (PPP); 2) to compare transforming growth factor beta 1 (TGF-β(1)) and platelet-derived growth factor isoform BB (PDGF-BB) concentrations in supernatants from pure PRG (P-PRG), platelet-poor gel (PPG), P-PRP lysate (positive control) and plasma (negative control); 3) to establish the possible correlations between all the studied cellular and molecular parameters. In all cases the three anticoagulants produced P-PRPs with significantly higher PLT counts compared with whole blood and PPP. The concentrations of WBCs were similar between P-PRP and whole blood, but significantly lower in PPP. The type of anticoagulant did not significantly affect the cell counts for each blood component. The anticoagulants also did not affect the MPV and PDW parameters. Independently of the anticoagulant used, all blood components presented significantly different concentrations of PDGF-BB and TGF-β(1). The highest growth factor (GF) concentrations were observed from P-PRP lysates, followed by PRG supernatants, PPP lysates, PPG supernatants and plasma. Significant correlations were observed between PLT and WBC counts (ρ = 0.80), PLT count and TGF-β(1) concentration (ρ = 0.85), PLT count and PDGF-BB concentration (ρ = 0.80) and PDGF-BB and TGF-β(1) concentrations (ρ = 0.75). The type of anticoagulant was not correlated with any of the variables evaluated. The anticoagulants did not

  9. Genetics Home Reference: gray platelet syndrome

    Science.gov (United States)

    ... disorder, platelet-type, 4 deficient alpha granule syndrome GPS grey platelet syndrome platelet alpha-granule deficiency platelet ... on PubMed Central Kahr WH, Hinckley J, Li L, Schwertz H, Christensen H, Rowley JW, Pluthero FG, ...

  10. Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

    Science.gov (United States)

    Buntru, Matthias; Vogel, Simon; Spiegel, Holger; Schillberg, Stefan

    2014-05-03

    Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

  11. Purification of human platelet-derived growth factor

    International Nuclear Information System (INIS)

    Raines, E.W.; Ross, R.

    1985-01-01

    The paper describes a method for purification of human platelet-derived growth factor (PDGF) from outdated platelet-rich plasma (PRP) using commonly available laboratory reagents and yielding a mitogen purified 800,000-fold over the starting material. [ 3 H]thymidine incorporation into DNA of cultured cells responsive to PDGF represents the most readily available method to follow its purification and define the biological activity of a purified preparation. Other assays to quantitate PDGF include radioreceptor assay and radioimmunoassay

  12. Platelet size does not correlate with platelet age.

    Science.gov (United States)

    Thompson, C B; Love, D G; Quinn, P G; Valeri, C R

    1983-08-01

    The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.

  13. Platelet size does not correlate with platelet age

    International Nuclear Information System (INIS)

    Thompson, C.B.; Love, D.G.; Quinn, P.G.; Valeri, C.R.

    1983-01-01

    The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75 Se-methionine, (2) in vitro labeling with 51 Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75 Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51 Cr and 14 C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation

  14. Reproducibility of Manual Platelet Estimation Following Automated Low Platelet Counts

    Directory of Open Access Journals (Sweden)

    Zainab S Al-Hosni

    2016-11-01

    Full Text Available Objectives: Manual platelet estimation is one of the methods used when automated platelet estimates are very low. However, the reproducibility of manual platelet estimation has not been adequately studied. We sought to assess the reproducibility of manual platelet estimation following automated low platelet counts and to evaluate the impact of the level of experience of the person counting on the reproducibility of manual platelet estimates. Methods: In this cross-sectional study, peripheral blood films of patients with platelet counts less than 100 × 109/L were retrieved and given to four raters to perform manual platelet estimation independently using a predefined method (average of platelet counts in 10 fields using 100× objective multiplied by 20. Data were analyzed using intraclass correlation coefficient (ICC as a method of reproducibility assessment. Results: The ICC across the four raters was 0.840, indicating excellent agreement. The median difference of the two most experienced raters was 0 (range: -64 to 78. The level of platelet estimate by the least-experienced rater predicted the disagreement (p = 0.037. When assessing the difference between pairs of raters, there was no significant difference in the ICC (p = 0.420. Conclusions: The agreement between different raters using manual platelet estimation was excellent. Further confirmation is necessary, with a prospective study using a gold standard method of platelet counts.

  15. Metabolomic analysis of platelets during storage

    DEFF Research Database (Denmark)

    Paglia, Giuseppe; Sigurjónsson, Ólafur E; Rolfsson, Óttar

    2015-01-01

    BACKGROUND: Platelet concentrates (PCs) can be prepared using three methods: platelet (PLT)-rich plasma, apheresis, and buffy coat. The aim of this study was to obtain a comprehensive data set that describes metabolism of buffy coat-derived PLTs during storage and to compare it with a previously...... published parallel data set obtained for apheresis-derived PLTs. STUDY DESIGN AND METHODS: During storage we measured more than 150 variables in 8 PLT units, prepared by the buffy coat method. Samples were collected at seven different time points resulting in a data set containing more than 8000...... after their collection. The transition was evident in PLT produced by both production methods. Apheresis-derived PLTs showed a clearer phenotype of PLT activation during early days of storage. The activated phenotype of apheresis PLTs was accompanied by a higher metabolic activity, especially related...

  16. Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

    Science.gov (United States)

    Tohidnezhad, Mersedeh; Lammel, Justus; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

    2017-01-01

    Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo. PMID:28808357

  17. Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

    Directory of Open Access Journals (Sweden)

    Andreas Bayer

    2017-01-01

    Full Text Available Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs or their clinically related formulations (e.g., Vivostat PRF® came recently into the physicians’ focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10 and late (transglutaminase-1 and involucrin differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR- dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

  18. Platelet transfusion practice in a tertiary care hospital

    International Nuclear Information System (INIS)

    Rehman, Z.; Alam, M.

    2002-01-01

    Objective: Pakistan is a developing country where platelet concentrates are prepared and administered to patients in only a few large centres of the country. A study was designed for appraisal of the current situation and to review the progress made so far. Design: It was a prospective, non-interventional study. Place and duration of study: The study was conducted at PNS Shifa, Karachi from January, 1995 to December, 1998. Subjects and Methods: During this study 588 random donor platelet concentrates were transfused to 66 patients 148 occasions. Random donor platelet concentrates were prepared by fractionation of whole blood using triple blood collecting bags. Pre-transfusion and one hour posttransfusion platelet counts of the patients were done. The efficacy of the platelet transfusion was monitored by noting the clinical response as well as doing one hour posttransfusion corrected counts increment (CCI).Results: On 114 (77%) occasions platelets were transfused prophylactically and 34 (23%) times therapeutically to stop major bleeding episodes. The mean pre-transfusion platelet count varied from 15.5 x 10/sup 9/1 to 28.5 x 10/sup 9/l in different clinical conditions. On average, 4 random donor platelet concentrates were administered on each occasion. The best response was observed in patients of aplastic anaemia and worst in cases of disseminated intravascular coagulation (DIC). Conclusion: Platelet concentrates administration was inappropriate in significant number of patients, therefore, each hospital should form transfusion committee to review transfusion practices guidelines for blood components usage and compliance to these guidelines by the clinicians. (author)

  19. Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.

    Science.gov (United States)

    Krawczyk, Z; Wu, C

    1987-08-15

    We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.

  20. Detection of circulating tumor lysate-reactive CD4+ T cells in melanoma patients

    DEFF Research Database (Denmark)

    Ladekarl, Morten; Agger, Ralf; Fleischer, Charlotte C

    2004-01-01

    PURPOSE: We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4+ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which...... was found in 1 of 4 patients radically operated for localized disease, whereas no responders were seen among 7 healthy donors. The fraction of circulating lysate-activated T cells ranged from 0.0037% to 0.080% of the CD4+ population. A negative result of the assay was found occasionally, especially...... expresses high amounts of melanoma antigens. METHODS: Fresh peripheral blood was incubated with and without lysate for 6 h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69+/IFN-gamma+ cells in the CD4+ population, the response...

  1. Platelet aggregation following trauma

    DEFF Research Database (Denmark)

    Windeløv, Nis A; Sørensen, Anne M; Perner, Anders

    2014-01-01

    We aimed to elucidate platelet function in trauma patients, as it is pivotal for hemostasis yet remains scarcely investigated in this population. We conducted a prospective observational study of platelet aggregation capacity in 213 adult trauma patients on admission to an emergency department (ED...... severity score (ISS) was 17; 14 (7%) patients received 10 or more units of red blood cells in the ED (massive transfusion); 24 (11%) patients died within 28 days of trauma: 17 due to cerebral injuries, four due to exsanguination, and three from other causes. No significant association was found between...... aggregation response and ISS. Higher TRAP values were associated with death due to cerebral injuries (P 

  2. Assessment of canine autologous platelet-rich plasma produced with a commercial centrifugation and platelet recovery kit.

    Science.gov (United States)

    Frye, Chris W; Enders, Andrew; Brooks, Marjory B; Struble, Angela M; Wakshlag, Joseph J

    2016-01-01

    To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood. Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs. This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively. Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

  3. Microparticle counts in platelet-rich and platelet-free plasma, effect of centrifugation and sample-processing protocols.

    Science.gov (United States)

    Chandler, Wayne L

    2013-03-01

    This study provides the first estimates of microparticle numbers in platelet-rich plasma (PRP) from normal individuals, closer to in-vivo levels, using higher-resolution flow cytometry. We measured platelet (CD41+) and annexin V+ microparticles in fresh and frozen aliquots of PRP, platelet-poor plasma, platelet-free plasma (PFP), and microparticles isolated by high-speed centrifugation. PRP from healthy individuals contained 730,000/μl total microparticles based on light-scattering measurements. A median of 27,000/μl microparticles in PRP were of platelet origin and 120,000/μl annexin V+, and of these, 24,000/μl were dual-positive procoagulant platelet microparticles. Double centrifugation of PRP removed 99% of platelets, but also 80% of annexin V+ CD41+, 93% of annexin V+ CD41-, and 58% of annexin V- CD41+ microparticles. Loss of microparticles with centrifugation varied from individual to individual. Microparticle counts after isolation by centrifugation and double washing were not significantly different than counts in the original PFP sample, but lower than in PRP. Freeze-thawing of PFP had no effect on platelet microparticle counts, but slightly increased annexin V+, CD41- counts. Freeze-thawing of isolated washed microparticles resulted in a 30-50% increase in annexin V+ microparticles. PRP contains large numbers of cellular microparticles, including platelet and annexin V+ microparticles, which are lost to varying degrees when PRP is double centrifuged to remove platelets. Microparticles remaining in PFP can be recovered by high-speed centrifugation without loss compared to the original PFP sample. Freeze-thawing has variable effects on microparticle counts depending on the sample preparation used.

  4. Comparison between mixed lysate antigen and α-actinin antigen in ELISA for serodiagnosis of trichomoniasis.

    Science.gov (United States)

    Kim, Seung-Ryong; Kim, Jung-Hyun; Park, Soon-Jung; Lee, Hye-Yeon; Kim, Yong-Suk; Kim, Yu-Mi; Hong, Yeon-Chul; Ryu, Jae-Sook

    2015-10-01

    The aim of this study was to identify an antigen suitable for ELISA for serodiagnosis of Trichomonas vaginalis (T. vaginalis) infection. Mixed lysate antigen (Ag) from eight strains of T. vaginalis and recombinant α-actinin protein was compared. The sera of three groups were examined by ELISA: 73 women infected with trichomoniasis served as a positive control, 31 male volunteers as a negative control, and 424 women attending an outpatient health screening at Hanyang University Guri Hospital. Based on the cutoff optical density for each Ag obtained with a negative control, the serosensitivity of the mixed lysate Ag (79.5%) was significantly higher than that of the α-actinin (52.1%) in the 73 patients with trichomoniasis. The specificity using lysate Ag and α-actinin was 100% and 96.8%, respectively. On the other hand, the positivity rate in 424 outpatients was 39.2% and 11.8% with mixed lysate and α-actinin Ag, respectively. Taken together, mixed lysate Ag showed higher sensitivity and specificity than α-actinin. Therefore, mixed lysate may be a better Ag than α-actinin for ELISA for the diagnosis of trichomoniasis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Nanomolar concentrations of adrenaline induce platelet adhesion in vitro.

    Science.gov (United States)

    Eriksson, Andreas C; Whiss, Per A

    2013-01-01

    Adrenaline is a platelet activator having a resting plasma concentration of adrenaline in micromolar concentrations. This makes it difficult to estimate the relevance of in vitro data for the in vivo situation. The aim of this study was to investigate experimental conditions in vitro that could detect platelet effects of adrenaline in nanomolar concentrations. Platelet adhesion to albumin and collagen was evaluated with a static platelet adhesion assay. Our results show that 10 nmol/l adrenaline induced platelet adhesion to albumin in platelet-rich plasma (PRP) prepared at 140 × g, while 100 nmol/l was necessary in order to increase adhesion of platelets prepared at 220 × g. The mean platelet volume was increased after preparation at 140 × g, suggesting that large reactive platelets contributed to the increased adrenaline sensitivity. At optimal Mg(2+)-concentration, adhesion to collagen was increased by 10 nmol/l adrenaline irrespective of centrifugal force applied during PRP preparation. More specifically, we defined two populations where adhesion to collagen was increased by 10 nmol/l adrenaline either upon centrifugation at 140 × g but not 220 × g or vice versa. In some experiments, platelet adhesion to collagen was induced by 3 nmol/l adrenaline, which corresponds to concentrations achieved during stress in vivo. In summary, the static adhesion assay is able to detect platelet activating effects of adrenaline very close to physiological concentrations. This is rare for in vitro assays and motivates further research about adrenergic signalling in platelets.

  6. Blood platelet inventory management

    NARCIS (Netherlands)

    Haijema, R.; van Dijk, N. M.; van der Wal, J.; Boucherie, Richard J.; van Dijk, Nico M.

    2017-01-01

    This paper illustrates how MDP or Stochastic Dynamic Programming (SDP) can be used in practice for blood management at blood banks; both to set regular production quantities for perishable blood products (platelets) and how to do so in irregular periods (as holidays). The state space is too large to

  7. Alternatives to allogeneic platelet transfusion.

    Science.gov (United States)

    Desborough, Michael J R; Smethurst, Peter A; Estcourt, Lise J; Stanworth, Simon J

    2016-11-01

    Allogeneic platelet transfusions are widely used for the prevention and treatment of bleeding in thrombocytopenia. Recent evidence suggests platelet transfusions have limited efficacy and are associated with uncertain immunomodulatory risks and concerns about viral or bacterial transmission. Alternatives to transfusion are a well-recognised tenet of Patient Blood Management, but there has been less focus on different strategies to reduce bleeding risk by comparison to platelet transfusion. Direct alternatives to platelet transfusion include agents to stimulate endogenous platelet production (thrombopoietin mimetics), optimising platelet adhesion to endothelium by treating anaemia or increasing von Willebrand factor levels (desmopressin), increasing formation of cross-linked fibrinogen (activated recombinant factor VII, fibrinogen concentrate or recombinant factor XIII), decreasing fibrinolysis (tranexamic acid or epsilon aminocaproic acid) or using artificial or modified platelets (cryopreserved platelets, lyophilised platelets, haemostatic particles, liposomes, engineered nanoparticles or infusible platelet membranes). The evidence base to support the use of these alternatives is variable, but an area of active research. Much of the current randomised controlled trial focus is on evaluation of the use of thrombopoietin mimetics and anti-fibrinolytics. It is also recognised that one alternative strategy to platelet transfusion is choosing not to transfuse at all. © 2016 John Wiley & Sons Ltd.

  8. Are adipose-derived stem cells cultivated in human platelet lysate suitable for heart valve tissue engineering?

    NARCIS (Netherlands)

    Frese, L.; Sasse, T.; Sanders, B.; Baaijens, F.P.T.; Beer, G.M.; Hoerstrup, S.P.

    2017-01-01

    Tissue-engineered heart valves represent a promising strategy for the growing need for valve replacements in cardiovascular medicine. Recent studies have shown that adipose-derived stem cells (ADSC) are a viable cell source, as they are readily available in both the young and the elderly, show

  9. Influência da coleta, da produção e da estocagem na qualidade dos concentrados de plaquetas Influence of collection, preparation and storage on the quality of platelet concentrates

    Directory of Open Access Journals (Sweden)

    Maria Aparecida V. Tostes

    2008-10-01

    Full Text Available Como o controle de qualidade dos concentrados de plaquetas (CP, feito na data de seu vencimento, não possibilita distinguir o momento e o procedimento que determina a eventual redução de sua qualidade, decidimos investigar, separadamente, a influência da coleta, da produção e da estocagem sobre a qualidade deste hemocomponente. Foram avaliados, em 33 CP randômicos, diariamente, durante cinco dias, os seguintes parâmetros: a agregação, o número de plaquetas e leucócitos, a pO2 e pCO2, o pH, sódio e potássio, a presença de swirling, grumos, hemácias e lipemia e cultura para bactérias. Observamos maior queda da agregação plaquetária com pares de agonistas durante a produção dos CP (de 99,4% para 59,8%, pAs quality control of platelet concentrates obtained at the expiration date does not distinguish between the different stages that may cause reductions in quality, such as collection and storage, we decided to separately investigate the influence of collection, processing and storage on the quality of these blood components. This study evaluated 33 random platelet concentrates daily for five days for the following parameters: aggregation, number of platelets and leukocytes, PO2 and PCO2, pH, sodium and potassium, the presence of swirling, platelet clots, red blood cells and lipemia, and bacteria culture. We observed a greater decrease in platelet aggregation with pairs of agonists during the production of platelet concentrates (from 99.4% to 59.8%, p<0.05, followed by a gradual drop during storage reaching 40.4% on day five. During storage the following were also observed: 1. a gradual drop of platelet concentration (p<0.05 although the values always remained higher than 5x1010/70 mL; 2. a decrease in leukocyte concentration (p<0.05; 3. increases in pO2 and decreases in pCO2 (p<0.05; 4. increase in the pH (p<0.05 from day 4 and in concentrations of sodium and potassium (in general p<0.05; 5. swirling in all platelet

  10. The influence of environmental variables on platelet concentration in horse platelet-rich plasma.

    Science.gov (United States)

    Rinnovati, Riccardo; Romagnoli, Noemi; Gentilini, Fabio; Lambertini, Carlotta; Spadari, Alessandro

    2016-07-04

    Platelet-rich plasma (PRP) commonly refers to blood products which contain a higher platelet (PLT) concentration as compared to normal plasma. Autologous PRP has been shown to be safe and effective in promoting the natural processes of soft tissue healing or reconstruction in humans and horses. Variability in PLT concentration has been observed in practice between PRP preparations from different patients or from the same individual under different conditions. A change in PLT concentration could modify PRP efficacy in routine applications. The aim of this study was to test the influence of environmental, individual and agonistic variables on the PLT concentration of PRP in horses. Six healthy Standardbred mares were exposed to six different variables with a one-week washout period between variables, and PRP was subsequently obtained from each horse. The variables were time of withdrawal during the day (morning/evening), hydration status (overhydration/dehydration) treatment with anti-inflammatory drugs and training periods on a treadmill. The platelet concentration was significantly higher in horses treated with a non-steroidal anti-inflammatory drug (P = 0.03). The leukocyte concentration increased 2-9 fold with respect to whole blood in the PRP which was obtained after exposure to all the variable considered. Environmental variation in platelet concentration should be taken into consideration during PRP preparation.

  11. Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols.

    Science.gov (United States)

    Jiménez-Aristizabal, Román F; López, Catalina; Álvarez, María E; Giraldo, Carlos; Prades, Marta; Carmona, Jorge U

    2017-09-01

    To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β 1 ) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs were measured by ELISA at 1h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p=0.01) effect of time was noted. All cytokines were detected after 6h of PRF clot culture until day 21. GF were detected at 1h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p=0.01) correlation was noticed between all the proteins evaluated. Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-β 1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

    Science.gov (United States)

    Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

    2011-08-18

    Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  13. Autologous Dendritic Cells Pulsed with Allogeneic Tumor Cell Lysate in Mesothelioma: From Mouse to Human.

    Science.gov (United States)

    Aerts, Joachim G J V; de Goeje, Pauline L; Cornelissen, Robin; Kaijen-Lambers, Margaretha E H; Bezemer, Koen; van der Leest, Cor H; Mahaweni, Niken M; Kunert, André; Eskens, Ferry A L M; Waasdorp, Cynthia; Braakman, Eric; van der Holt, Bronno; Vulto, Arnold G; Hendriks, Rudi W; Hegmans, Joost P J J; Hoogsteden, Henk C

    2018-02-15

    Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate-pulsed DC immunotherapy is effective in mice and safe in humans. Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte-derived DCs pulsed with tumor lysate from five mesothelioma cell lines. Results: In mice, allogeneic lysate-pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1-20.3] and median OS not reached (median follow-up = 22.8 months). Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766-76. ©2017 AACR . ©2017 American Association for Cancer Research.

  14. Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

    Directory of Open Access Journals (Sweden)

    Monique Ramos de Oliveira Trugilho

    2017-05-01

    Full Text Available Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet

  15. The Role of a Platelet Lysate-Based Compartmentalized System as a Carrier of Cells and Platelet-Origin Cytokines for Periodontal Tissue Regeneration

    NARCIS (Netherlands)

    Babo, P.S.; Cai, X.; Plachokova, A.S.; Reis, R.L.; Jansen, J.A.; Gomes, M.E.; Walboomers, X.F.

    2016-01-01

    Currently available clinical therapies are not capable to regenerate tissues that are lost by periodontitis. Tissue engineering can be applied as a strategy to regenerate reliably the tissues and function of damaged periodontium. A prerequisite for this regeneration is the colonization of the defect

  16. Effects of hormones on platelet aggregation.

    Science.gov (United States)

    Farré, Antonio López; Modrego, Javier; Zamorano-León, José J

    2014-04-01

    Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

  17. Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

    International Nuclear Information System (INIS)

    Kornecki, E.; Ehrlich, Y.H.; Egbring, R.; Gramse, M.; Seitz, R.; Eckardt, A.; Lukasiewicz, H.; Niewiarowski, S.

    1988-01-01

    The authors have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125 I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase. Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen-binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125 I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. They conclude that (1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and (2) human granulocyte elastase exposes active fibrongen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen

  18. Human Platelet Senescence Study.

    Science.gov (United States)

    1980-03-01

    ability to measure certain enzymes to their oxidation-reduc other enzymes which can be measured by o phosphatase , acid phosphatase , chymotryp...alkaline sin, trypsin, esterases (17)); M use of n A or wheat germ agglutinin in the second etect specific carbohydrate constituents. We have...Von Willebrand factor. Nurden and Caen also demonstrated that GPI was rich in sialic acid (5) and probably responsible for the platelets’ surface

  19. The effect of lysate of spleen cells after low dose radiation (LDR) on NK activity

    International Nuclear Information System (INIS)

    Lu Duicai; Su Liaoyuan

    2003-01-01

    To find effect of lysate of spleen cells after LDR on NK activity of CD 57 cells or non-CD 57 cells, lysate of spleen cells after LDR were extracted. McAb (anti CD 57 cells) was used to separate CD 57 cells from human peripheral blood by Panning direct method. The CD 57 cells and non-CD 57 cells were used as effective cells. K 562 cells labelled by 3 H-TdR were used as target cells. The ratio of effect cells to target cells was 10:1. NK activity of CD 57 cells or non-CD 5 -7 cells with the lysate of spleen cells after LDR was reflected by the efficiency of anti tumor cells. The 3 H-TdR incorporation in K 562 cells cultured with non-CD 57 cells was significantly lower than that with CD 57 cells. After use of the lysate of spleen cells after LDR, NK activities of CD 57 cells and non-CD 57 cells were 1.24 and 1.58 respectively. They were both increased obviously compared with control groups. The effect of anti K 562 cells of non-CD 57 cells is even greater than that of CD 57 cells. The lysate of spleen cells after LDR can enhance the effect of both non-CD 57 cells and CD 57 cells

  20. Platelet concentration of plateletrich plasma from dogs, obtained through three centrifugation speeds

    Directory of Open Access Journals (Sweden)

    Vanessa Couto de Magalhães Ferraz

    2007-12-01

    Full Text Available The platelets release at least 4 growth factors (Platelet Derived Growth Factor. ²1 and ²2 Transforming Growth Factors and Insulin-like Growth Factor which are responsible for the migration and activation of cells that will start the reparation of soft tissues and bones. The Platelet Rich Plasma is an autogenous source for Growth Factors, obtained by platelet concentration by centrifuging total blood. This study aimed the comparison of platelet concentrations in plasma centrifuged in three different centrifugation speeds (1300, 1600 e 3200rpm, for the production of platelet rich plasma. Blood was drowned from 15 dogs, 40ml of each, and these were divided into four groups and centrifuged at 800rpm. Then the first group was centrifuged at 1300rpm, the second at 1600rpm, the third at 3200rpm and the last was used as control, named plasma. The mean percentage increase in the platelet concentration for each technique was: 1300 - 183%, 1600 - 210% and 3200 - 222%. But in centrifugation at 3200 rpm, platelets presented altered morphology and different sizes in every sample studied, which was understood as severe cell damage. It was concluded that the best technique for the preparation of the platelet rich plasma in dogs consisted of the previous centrifugation of the blood at 800rpm for ten minutes, and then the plasma should be separated. This plasma is then submitted to a second centrifugation of 1600rpm for 10 minutes, and the platelet poor plasma is separated and discharged.

  1. Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

    Directory of Open Access Journals (Sweden)

    Chiang Cheryl L-L

    2011-11-01

    Full Text Available Abstract Background Dendritic cells (DCs are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines. Methods Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1 culture media; 2 culture surface; 3 duration of activating DCs with lipopolysaccharide (LPS and interferon (IFN-gamma; 4 method of DC harvest; and 5 cryomedia and final DC product formulation. Results DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning® tissue-culture treated surface and Corning® ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs, and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and

  2. Superoxide dismutase of human platelets

    International Nuclear Information System (INIS)

    Kimura, Akiro; Fujimura, Kingo; Kuramoto, Atsushi

    1979-01-01

    Superoxide dismutase (S.O.D.) is the enzyme to protect from destructive effect of superoxide (O 2 -) produced in many metabolic pathways related to oxygen. The purpose of this study was to investigate the possibility that S.O.D. may play an important role in the platelet function. The cytoplasmic and mitochondrial S.O.D. has been investigated spectrophotometrically and gel electrophoretically in human platelets from eleven patients of chronic myelogenous leukemia (CML) and three patients of primary thrombocythemia (P.Th.). Neither deficiency nor abnormality of cytoplasmic and mitochondrial S.O.D. has been found electrophoretically in any case compared to normal platelets. However, the total activity from three of the CML patients and one of the P.Th. patients were above 3 unit/mg platelet protein (normal subject: 2.11 - 2.70 unit/mg protein), suggesting the possibility either that more O 2 -production occurs in the platelets or that rather little O 2 -production due to much O 2 -deprivation by the increased S.O.D. The S.O.D. activity of human platelets has been also investigated in several conditions, where much O 2 -generation might occur in platelets. Sodium fluoride (2 mM), which increases platelet O 2 -production about 3 fold, had no effect on platelet S.O.D. The aggregated platelets induced by ADP (10 -5 M), epinephrin (50 μg/ml), ristocetin (1.5 mg/ml) or collagen (1 - 20 μg/ml) had no increase of S.O.D. activity compared to that from non aggregated platelets. X-ray irradiation (1,000 - 20,000R) had not induced its activity increase or decrease. These findings indicated the induction of platelet S.O.D. was not brought about under these conditions. (author)

  3. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2012-07-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  4. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    International Nuclear Information System (INIS)

    Perçin, Işık; Karakoç, Veyis; Akgöl, Sinan; Aksöz, Erol; Denizli, Adil

    2012-01-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m 2 /g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 μg/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: ► Magnetic nanoparticles have several advantages over conventional adsorbents. ► MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. ► pDNA adsorption amount was 154 mg/g. ► Fifty-fold capacity increase was obtained when compared to conventional matrices.

  5. Platelet-released growth factors inhibit proliferation of primary keratinocytes in vitro.

    Science.gov (United States)

    Bayer, Andreas; Tohidnezhad, Mersedeh; Berndt, Rouven; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Simanski, Maren; Gläser, Regine; Harder, Jürgen

    2018-01-01

    Autologous thrombocyte concentrate lysates as platelet-released growth factors (PRGF) or Vivostat Platelet Rich Fibrin (PRF ® ) represent important tools in modern wound therapy, especially in the treatment of chronic, hard-to-heal or infected wounds. Nevertheless, underlying cellular and molecular mechanisms of the beneficial clinical effects of a local wound therapy with autologous thrombocyte concentrate lysates are poorly understood. Recently, we have demonstrated that PRGF induces antimicrobial peptides in primary keratinocytes and accelerates keratinocytes' differentiation. In the present study we analyzed the influence of PRGF on primary human keratinocytes' proliferation. Using the molecular proliferation marker Ki-67 we observed a concentration- and time dependent inhibition of Ki-67 gene expression in PRGF treated primary keratinocytes. These effects were independent from the EGFR- and the IL-6-R pathway. Inhibition of primary keratinocytes' proliferation by PRGF treatment was confirmed in colorimetric cell proliferation assays. Together, these data indicate that the clinically observed positive effects of autologous thrombocytes concentrates in the treatment of chronic, hard-to-heal wounds are not based on an increased keratinocytes proliferation. Copyright © 2017 Elsevier GmbH. All rights reserved.

  6. Overview of platelet physiology and laboratory evaluation of platelet function.

    Science.gov (United States)

    Rodgers, G M

    1999-06-01

    Appropriate laboratory testing for the platelet-type bleeding disorders hinges on an adequate assessment in the history and physical examination. Patients with histories and screening laboratory results consistent with coagulation disorders (hemophilia, disseminated intravascular coagulation) are not appropriate candidates for platelet function testing. In contrast, patients with a lifelong history of platelet-type bleeding symptoms and perhaps a positive family history of bleeding would be appropriate for testing. Figure 6 depicts one strategy to evaluate these patients. Platelet morphology can easily be evaluated to screen for two uncommon qualitative platelet disorders: Bernard-Soulier syndrome (associated with giant platelets) and gray platelet syndrome, a subtype of storage pool disorder in which platelet granulation is morphologically abnormal by light microscopy. If the bleeding disorder occurred later in life (no bleeding with surgery or trauma early in life), the focus should be on acquired disorders of platelet function. For those patients thought to have an inherited disorder, testing for vWD should be done initially because approximately 1% of the population has vWD. The complete vWD panel (factor VIII coagulant activity, vWf antigen, ristocetin cofactor activity) should be performed because many patients will have abnormalities of only one particular panel component. Patients diagnosed with vWD should be classified using multimeric analysis to identify the type 1 vWD patients likely to respond to DDAVP. If vWD studies are normal, platelet aggregation testing should be performed, ensuring that no antiplatelet medications have been ingested at least 1 week before testing. If platelet aggregation tests are normal and if suspicion for an inherited disorder remains high, vWD testing should be repeated. The evaluation of thrombocytopenia may require bone marrow examination to exclude primary hematologic disorders. If future studies with thrombopoietin assays

  7. 111In,113In-labelled platelets. I

    International Nuclear Information System (INIS)

    Komarek, P.; Poledne, R.; Charvat, J.; Konopkova, M.; Komarkova, I.

    1983-01-01

    111 In-8-hydroxyquinoline (oxine) is used for labelling blood platelets which serves diagnostic purposes in medicine. For the preparation of the complex of radioactive indium with oxine, 111 In and sup(113m)In were used under optimal conditions. Two different preparation methods for the labelled complex are described. Both methods studied the effect of pH, the amount of chelate agents and the mode of filtration on the yields of the labelled oxine. The blood platelets labelling is influenced not only by the quality of labelled oxine but also by their number. The organ distribution in laboratory animals proved that platelets were not impaired during separation and labelling and that they were suitable for diagnostic use. (author)

  8. Subpopulations in purified platelets adhering on glass.

    Science.gov (United States)

    Donati, Alessia; Gupta, Swati; Reviakine, Ilya

    2016-06-22

    Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process.

  9. Do methodological differences account for the current controversy on tissue factor expression in platelets?

    Science.gov (United States)

    Brambilla, Marta; Rossetti, Laura; Zara, Chiara; Canzano, Paola; Giesen, Peter L A; Tremoli, Elena; Camera, Marina

    2018-06-01

    Tissue factor (TF), the key activator of the blood coagulation cascade and of thrombus formation, is also expressed by circulating human platelets. Despite the documented in-depth characterization of platelet TF carried out in the past 15 years, some authors still fail to identify TF in platelets, especially when assessment in platelet-rich plasma (PRP) or washed platelets is carried out. This study aims to extend the characterization of the subset of TF-positive platelets in PRP from healthy subjects and to verify how different centrifugation forces, used to prepare the PRP, could affect the analysis of TF-positive platelets. Data indicate that large-size platelets express significantly higher amount of TF compared to small-size cells, in terms of both TF protein and TF mRNA. Upon stimulation, large platelets readily expose on the cell membrane TF, which is functionally active, i.e., able to generate factor Xa (FXa) as well as thrombin. By contrast, TF activity in small platelets is almost completely quenched by tissue factor pathway inhibitor (TFPI), becoming indeed detectable only after treatment with an anti-TFPI antibody. Our data highlight that particular attention must be paid to the preparation and collection of the PRP since such preanalytical variables may influence the platelet recovery and in turn affect subsequent analysis, whether it is flow cytometry, functional activity tests, proteome, or transcriptome analysis. Indeed, the TF-positive subset of large platelets can easily be lost if centrifugation protocols are not optimized, thus erroneously leading to a false-negative result.

  10. One size doesn’t fit all: Should we reconsider the introduction of cold-stored platelets in blood bank inventories? [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Alessandra Berzuini

    2017-02-01

    Full Text Available Platelet concentrates are universally prepared with a standard method and stored for 5 days at room temperature (20–24°C in gentle agitation. Currently, there is a renewed interest in the possibility of storing platelet concentrates below the standard temperatures. In fact, cold platelets might be more effective in bleeding patients and have a lower risk of bacterial transmission. Inventories including platelets at different temperatures may favour patient-centred strategies for prophylactic or therapeutic transfusions.

  11. Differential Expression Analysis by RNA-Seq Reveals Perturbations in the Platelet mRNA Transcriptome Triggered by Pathogen Reduction Systems

    OpenAIRE

    Osman, Abdimajid; Hitzler, Walter E.; Ameur, Adam; Provost, Patrick

    2015-01-01

    Platelet concentrates (PCs) are prepared at blood banks for transfusion to patients in certain clinical conditions associated with a low platelet count. To prevent transfusion-transmitted infections via PCs, different pathogen reduction (PR) systems have been developed that inactivate the nucleic acids of contaminating pathogens by chemical cross-linking, a mechanism that may also affect platelets' nucleic acids. We previously reported that treatment of stored platelets with the PR system Int...

  12. Ultrastructure and growth factor content of equine platelet-rich fibrin gels.

    Science.gov (United States)

    Textor, Jamie A; Murphy, Kaitlin C; Leach, J Kent; Tablin, Fern

    2014-04-01

    To compare fiber diameter, pore area, compressive stiffness, gelation properties, and selected growth factor content of platelet-rich fibrin gels (PRFGs) and conventional fibrin gels (FGs). PRFGs and conventional FGs prepared from the blood of 10 healthy horses. Autologous fibrinogen was used to form conventional FGs. The PRFGs were formed from autologous platelet-rich plasma of various platelet concentrations (100 × 10³ platelets/μL, 250 × 10³ platelets/μL, 500 × 10³ platelets/μL, and 1,000 × 10³ platelets/μL). All gels contained an identical fibrinogen concentration (20 mg/mL). Fiber diameter and pore area were evaluated with scanning electron microscopy. Maximum gelation rate was assessed with spectrophotometry, and gel stiffness was determined by measuring the compressive modulus. Gel weights were measured serially over 14 days as an index of contraction (volume loss). Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were quantified with ELISAs. Fiber diameters were significantly larger and mean pore areas were significantly smaller in PRFGs than in conventional FGs. Gel weight decreased significantly over time, differed significantly between PRFGs and conventional FGs, and was significantly correlated with platelet concentration. Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were highest in gels and releasates derived from 1,000 × 10³ platelets/μL. The inclusion of platelets in FGs altered the architecture and increased the growth factor content of the resulting scaffold. Platelets may represent a useful means of modifying these gels for applications in veterinary and human regenerative medicine.

  13. Antagonism of Sorafenib and Regorafenib actions by platelet factors in hepatocellular carcinoma cell lines

    International Nuclear Information System (INIS)

    D’Alessandro, Rosalba; Refolo, Maria G; Lippolis, Catia; Giannuzzi, Grazia; Carella, Nicola; Messa, Caterina; Cavallini, Aldo; Carr, Brian I

    2014-01-01

    Platelets are frequently altered in hepatocellular carcinoma (HCC) patients. Platelet lysates (hPL) can enhance HCC cell growth and decrease apoptosis. The aims were to evaluate whether hPL can modulate the actions of Sorafenib or Regorafenib, two clinical HCC multikinase antagonists. Several human HCC cell lines were grown in the presence and absence of Sorafenib or Regorafenib, with or without hPL. Growth was measured by MTT assay, apoptosis was assessed by Annexin V and by western blot, and autophagy and MAPK growth signaling were also measured by western blot, and migration and invasion were measured by standard in vitro assays. Both Sorafenib and Regorafenib-mediated inhibition of cell growth, migration and invasion were all antagonized by hPL. Drug-mediated apoptosis and decrease in phospho-ERK levels were both blocked by hPL, which also increased anti-apoptotic phospho-STAT, Bax and Bcl-xL levels. Preliminary data, obtained with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), included in hPL, revealed that these factors were able to antagonized Sorafenib in a proliferation assay, in particular when used in combination. Platelet factors can antagonize Sorafenib or Regorafenib-mediated growth inhibition and apoptosis in HCC cells. The modulation of platelet activity or numbers has the potential to enhance multikinase drug actions

  14. Influence of Oxidative Stress on Stored Platelets

    OpenAIRE

    K. Manasa; R. Vani

    2016-01-01

    Platelet storage and its availability for transfusion are limited to 5-6 days. Oxidative stress (OS) is one of the causes for reduced efficacy and shelf-life of platelets. The studies on platelet storage have focused on improving the storage conditions by altering platelet storage solutions, temperature, and materials. Nevertheless, the role of OS on platelet survival during storage is still unclear. Hence, this study was conducted to investigate the influence of storage on platelets. Platele...

  15. Platelet Concentrates: Past, Present and Future

    OpenAIRE

    Prakash, Shobha; Thakur, Aditi

    2011-01-01

    Platelets play a crucial role in hemostasis and wound healing, platelet growth factors are well known source of healing cytokines. Numerous techniques of autologous platelet concentrates have been developed and applied in oral and maxillofacial surgery. This review describes the evolution of the first and second generation of platelet concentrates (platelet rich plasma and platelet rich fibrin respectively) from their fore runner-fibrin sealants.

  16. Assessment of Platelet Profile of Healthy Volunteers in the ...

    African Journals Online (AJOL)

    ADOWIE PERE

    A similar pattern was observed for Mean Platelet Volume (MPV). However, Platelet ... Keywords: Platelet Count, Plateletcrit, Mean Platelet Volume, Platelet Distribution Width, Trimesters, ... bleeding disorders, diabetes and drugs capable of.

  17. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

    Science.gov (United States)

    Frelinger, A L; Torres, A S; Caiafa, A; Morton, C A; Berny-Lang, M A; Gerrits, A J; Carmichael, S L; Neculaes, V B; Michelson, A D

    2016-01-01

    Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

  18. Immune Response Generated With the Administration of Autologous Dendritic Cells Pulsed With an Allogenic Tumoral Cell-Lines Lysate in Patients With Newly Diagnosed Diffuse Intrinsic Pontine Glioma

    Directory of Open Access Journals (Sweden)

    Daniel Benitez-Ribas

    2018-04-01

    Full Text Available Background and objectiveDiffuse intrinsic pontine glioma (DIPG is a lethal brainstem tumor in children. Dendritic cells (DCs have T-cell stimulatory capacity and, therefore, potential antitumor activity for disease control. DCs vaccines have been shown to reactivate tumor-specific T cells in both clinical and preclinical settings. We designed a phase Ib immunotherapy (IT clinical trial with the use of autologous dendritic cells (ADCs pulsed with an allogeneic tumors cell-lines lysate in patients with newly diagnosed DIPG after irradiation (radiation therapy.MethodsNine patients with newly diagnosed DIPG met enrollment criteria. Autologous dendritic cell vaccines (ADCV were prepared from monocytes obtained by leukapheresis. Five ADCV doses were administered intradermally during induction phase. In the absence of tumor progression, patients received three boosts of tumor lysate every 3 months during the maintenance phase.ResultsVaccine fabrication was feasible in all patients included in the study. Non-specific KLH (9/9 patients and specific (8/9 patients antitumor response was identified by immunologic studies in peripheral blood mononuclear cells (PBMC. Immunological responses were also confirmed in the T lymphocytes isolated from the cerebrospinal fluid (CSF of two patients. Vaccine administration resulted safe in all patients treated with this schema.ConclusionThese preliminary results demonstrate that ADCV preparation is feasible, safe, and generate a DIPG-specific immune response detected in PBMC and CSF. This strategy shows a promising backbone for future schemas of combination IT.

  19. Impact of local anaesthetics and needle calibres used for painless PRP injections on platelet functionality.

    Science.gov (United States)

    Bausset, Olivier; Magalon, Jeremy; Giraudo, Laurent; Louis, Marie-Laure; Serratrice, Nicolas; Frere, Corrine; Magalon, Guy; Dignat-George, Françoise; Sabatier, Florence

    2014-01-01

    The platelet-rich plasma (PRP) is an autologous biotherapy commonly used for its healing properties. Once activated, platelets released a real "cocktail" of growth factor and cytokines implied in numerous regenerative processes. However the impact of medical practices associated to PRP therapeutic use on platelets functionality remains poorly known. we evaluated the in vitro effects of two commonly used local anesthetics (Xylocaine(*) and Naropin(*)) on PRP functionality. We also investigated the quantity and quality of PRP that passed through the smallest gauge needle commercialized. PRP from 9 healthy volunteers were prepared using our previously described home made purification protocol. Platelet aggregation capacity was evaluated by aggregometry assays and the growth factor release was determined by ELISA after platelet activation. We also evaluated the platelet activation status, reactivity and stability of platelets by flow cytometry using the P-selectin expression marker. the association of local anaesthetics with PRP injections resulted in a significant decrease of platelets functionality, assessed by their capacity of aggregating. Local anaesthetics did not interfere with the growth factor release. The different needle sizes and calibres tested for PRP injections did not influence the platelet functionality. the use of local anaesthetics to prevent pain during PRP injections could compromise the therapeutic potential of PRP. These results suggest using carefully local anaesthetics or limiting their use as often is possible. To minimize injection pain, we recommend using 30 G needles. These data will lead to clinical recommendations for painless and controlled PRP injections.

  20. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    Science.gov (United States)

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-11-05

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

  1. Mean platelet volume and mean platelet volume/platelet count ratio

    African Journals Online (AJOL)

    Amira M. Elsayed

    2016-03-30

    Mar 30, 2016 ... The aim of this study was to compare the MPV and mean platelet volume/platelet count ... brain stroke, both in the acute phase and long after disease.17 ... males, while the healthy controls comprised 12 females and 8.

  2. Encapsulation of Platelet in Kefiran Polymer and Detection of Bioavailability of Immobilized Platelet in Probiotic Kefiran as A New Drug for Surface Bleeding

    Directory of Open Access Journals (Sweden)

    Anahita Jenab

    2015-11-01

    Full Text Available Background : Kefir contains lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc, Acetobacter and Streptococcus and yeasts (Kluyveromyces, Torula, Candida, Saccharomyces .Kefiran is the polysaccharide produced by lactic acid bacteria in kefir.Methods : Kefiran was prepared from milk containing 0.5% fat and 10 grams kefir grains and was separated from kefir by ethanol (0.02 gram following entrapping the platelets to this polymer. Ligand of the platelet-polysaccharide was studied by FTIR.Results : FTIR results showed that the bands of C-O and C-O-C connections were formed and the polysaccharides had been attached to the receptors of the platelet glycoproteins (GP Ib,GPIIb / IIIa. Stability and encapsulation of the platelet and kefiran were assessed by Coulter Counter. Encapsulation of the platelets by polysaccharide at the beginning caused to reduce the number of platelets following by releasing of 50% of the platelets after 3 hours.Conclusion : The platelets were encapsulated in kefiran polymer and detected for bioavailability as new drug for surface bleeding. Also, kefiran has antimicrobial and antifungal properties. On the other hand, the existence of nisin in kefiran could be useful as an antibacterial lantibiotic. 

  3. Platelet biomechanics, platelet bioenergetics, and applications to clinical practice and translational research.

    Science.gov (United States)

    George, Mitchell J; Bynum, James; Nair, Prajeeda; Cap, Andrew P; Wade, Charles E; Cox, Charles S; Gill, Brijesh S

    2018-07-01

    The purpose of this review is to explore the relationship between platelet bioenergetics and biomechanics and how this relationship affects the clinical interpretation of platelet function devices. Recent experimental and technological advances highlight platelet bioenergetics and biomechanics as alternative avenues for collecting clinically relevant data. Platelet bioenergetics drive energy production for key biomechanical processes like adhesion, spreading, aggregation, and contraction. Platelet function devices like thromboelastography, thromboelastometry, and aggregometry measure these biomechanical processes. Platelet storage, stroke, sepsis, trauma, or the activity of antiplatelet drugs alters measures of platelet function. However, the specific mechanisms governing these alterations in platelet function and how they relate to platelet bioenergetics are still under investigation.

  4. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

    Roth, G.J.; Machuga, E.T.

    1982-01-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

  5. Platelet Glycoprotein Ib-IX and Malignancy

    Science.gov (United States)

    2010-09-01

    provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

  6. Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

    Science.gov (United States)

    Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

    2000-09-01

    Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

  7. Platelet Rich Plasma- mechanism of action and clinical applications

    Directory of Open Access Journals (Sweden)

    Cristina N. Cozma

    2016-11-01

    Full Text Available Platelet-rich plasma (PRP is a blood-derived fraction containing high level of platelets, a high concentration of leukocytes and growth factors. PRP therapy has been growing as a viable treatment alternative for a number of clinical applications and has a potential benefit for use in wound healing. Nowadays platelet rich plasma is used in stimulating wound healing in skin and soft tissue ulcerations, accelerating wound healing in diabetic patients and facilitating bone proliferation in orthopedic and trauma surgery. It has also applications in maxillofacial surgery, spinal surgery, plastic and esthetic surgery, heart surgery and burns. This review of the literature shows a limited number of studies realized on humans that sustain PRP applications in orthopedic and plastic surgery. As the use of PRP increases, more properly structured clinical studies are necessary to confirm the results and to establish clearly the techniques of preparing, the conditions and the clinical indications of applying this therapy.

  8. Platelet-rich-fibrin: A novel root coverage approach

    Directory of Open Access Journals (Sweden)

    Anilkumar K

    2009-01-01

    Full Text Available Treatment of gingival recession has become an important therapeutic issue due to increasing cosmetic demand. Multiple surgical procedures have been developed to obtain predictable esthetic root coverage. More specifically, after periodontal regenerative surgery, the aim is to achieve complete wound healing and regeneration of the periodontal unit. A recent innovation in dentistry is the preparation and use of platelet-rich plasma (PRP, a concentrated suspension of the growth factors, found in platelets. These growth factors are involved in wound healing and postulated as promoters of tissue regeneration. This paper reports the use of PRF membrane for root coverage on the labial surfaces of the mandibular anterior teeth. This was accomplished using laterally displaced flap technique with platelet rich fibrin (PRF membrane at the recipient site.

  9. Prophylactic platelets in dengue

    DEFF Research Database (Denmark)

    Whitehorn, James; Rodriguez Roche, Rosmari; Guzman, Maria G

    2012-01-01

    Dengue is the most important arboviral infection of humans. Thrombocytopenia is frequently observed in the course of infection and haemorrhage may occur in severe disease. The degree of thrombocytopenia correlates with the severity of infection, and may contribute to the risk of haemorrhage...... of platelets in dengue. Respondents were all physicians involved with the treatment of patients with dengue. Respondents were asked that their answers reflected what they would do if they were the treating physician. We received responses from 306 physicians from 20 different countries. The heterogeneity...... of the responses highlights the variation in clinical practice and lack of an evidence base in this area and underscores the importance of prospective clinical trials to address this key question in the clinical management of patients with dengue....

  10. Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles

    NARCIS (Netherlands)

    Rank, A.; Nieuwland, R.; Liebhardt, S.; Iberer, M.; Grützner, S.; Toth, B.; Pihusch, R.

    2011-01-01

    Background and Objectives Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). Material and Methods MP were double

  11. Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations.

    Science.gov (United States)

    Hom, Sarah; Chen, Li; Wang, Tony; Ghebrehiwet, Berhane; Yin, Wei; Rubenstein, David A

    2016-11-01

    Tobacco smoke extracts prepared from both mainstream and sidestream smoking have been associated with heightened platelet activation, aggregation, adhesion, and inflammation. Conversely, it has been shown that pure nicotine inhibits similar platelet functions. In this work, we 1) evaluated the effects of e-cigarette extracts on platelet activities and 2) elucidated the differences between the nicotine-dependent and non-nicotine dependent (e.g. fine particulate matter or toxic compounds) effects of tobacco and e-cigarette products on platelet activities. To accomplish these goals, platelets from healthy volunteers (n = 50) were exposed to tobacco smoke extracts, e-cigarette vapor extracts, and pure nicotine and changes in platelet activation, adhesion, aggregation, and inflammation were evaluated, using optical aggregation, flow cytometry, and ELISA methods. Interestingly, the exposure of platelets to e-vapor extracts induced a significant up-regulation in the expression of the pro-inflammatory gC1qR and cC1qR and induced a marked increase in the deposition of C3b as compared with traditional tobacco smoke extracts. Similarly, platelet activation, as measured by a prothrombinase based assay, and platelet aggregation were also significantly enhanced after exposure to e-vapor extracts. Finally, platelet adhesion potential toward fibrinogen, von Willebrand factor, and other platelets was also enhanced after exposure to e-cigarette vapor extracts. In the presence of pure nicotine, platelet functions were observed to be inhibited, which further suggests that other constituents of tobacco smoke and electronic vapor can antagonize platelet functions, however, the presence of nicotine in extracts somewhat perpetuated the platelet functional changes in a dose-dependent manner.

  12. Platelet-rich plasma and chronic wounds: remaining fibronectin may influence matrix remodeling and regeneration success.

    Science.gov (United States)

    Moroz, Andrei; Deffune, Elenice

    2013-11-01

    Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Recent reports were analyzed and presented as direct evidences of this hypotheses. Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. Platelet-rich fibrin in the treatment of periodontal bone defects.

    Science.gov (United States)

    Ranganathan, Aravindhan T; Chandran, Chitraa R

    2014-05-01

    Periodontitis is characterized by the formation of true pockets, bone loss and attachment loss. Various techniques have been attempted in the past to truly regenerate the lost periodontal structures, albeit with variable outcome. In this evolution, the technique being tried out widely is the use of platelet rich concentrates, namely platelet-rich fibrin (PRF). In this report, we present a case of surgical treatment of osseous bone defects namely two walled crater and dehiscence treated in posterior teeth with autologously prepared platelet rich fibrin mixed with hydroxy apatite bone graft and PRF in the form of a membrane. Our results showed clinical improvements in all the clinical parameters postoperatively namely the pocket depth reduction and gain in attachment level and hence, PRF can be used alone or in combination with the bone graft to yield successful clinical results in treating periodontal osseous defects. Platelet-rich fibrin is an effective alternative to platelet-rich plasma (PRP) in reconstructing bone defects.

  14. The use of autologous platelet-leukocyte gels to enhance the healing process in surgery, a review

    NARCIS (Netherlands)

    Everts, P. A.; Overdevest, E. P.; Jakimowicz, J. J.; Oosterbos, C. J.; Schonberger, J. P.; Knape, J. T.; van Zundert, A.

    2007-01-01

    Background: The therapeutic use of autologously prepared, platelet-leukocyte-enriched gel (PLG) is a relatively new technology for the stimulation and acceleration of soft tissue and bone healing. The effectiveness of this procedure lies in the delivery of a wide range of platelet growth factors

  15. Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo

    Directory of Open Access Journals (Sweden)

    Daniela eWeth

    2015-04-01

    Full Text Available At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P. It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (105/µl, 106/µl, 107/µl and assessed in mice with different genetic backgrounds (WT, S1P1fl/fl, SNS-S1P1-/-, S1P3-/-. Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralisation of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P.

  16. Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

    Science.gov (United States)

    Selheim, F; Holmsen, H; Vassbotn, F S

    1999-08-15

    We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

  17. Blood clotting and platelets: a delicate balance

    International Nuclear Information System (INIS)

    Heyns, A. du P.; Loetter, M.G.; Badenhorst, P.N.

    1988-01-01

    The Medical Research Council Blood Platelet Research Unit has studied many of the facets of platelets. The research highlights of the Unit include the following: the identification of ADPase, an enzyme found in the blood vessel wall, which breaks down adenosine diphospate (ADP) to adenosine, and which inhibits platelet aggregation; the determination of the relationship between the biochemical activity of the vessel wall with the development of atherosclerosis; the development of a method to label platelets with the compound In-111-oxine; the use of labelled platelets to study platelets in the spleen: an investigation of the life-span of platelets, which revealed how aged platelets are removed from circulation, and recently a study of methods to demonstrate in vivo activation of platelets. 1 fig

  18. Combinations of SPR and MS for Characterizations of Native and Recombinant Proteins in Cell Lysates

    DEFF Research Database (Denmark)

    Borch, Jonas; Roepstorff, Peter

    2006-01-01

    Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response...... and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands...... of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein...

  19. The effect of sibutramine on platelet morphology of Spraque-Dawley rats fed a high energy diet.

    Science.gov (United States)

    Oberholzer, Hester Magdalena; Van Der Schoor, Ciska; Pretorius, Etheresia

    2013-06-01

    The aim of this study was to investigate the effect of Sibutramine on platelet ultrastructure and discuss the morphological observations in relation to known physiological effects of the compound. Six-week-old, female Spraque-Dawley rats were used in this study. The animals were placed on a high energy diet after which sibutramine administration followed. Blood was drawn on the day of termination and platelet rich plasma was obtained to prepare plasma smears for analysis. Scanning electron microscopy was used to investigate the ultrastructure of the platelets. Platelets of the Sibutramine-treated animals showed smooth surface with limited pseudopodia formation when compared with that of the control animals. Higher magnification of the platelet surface showed membrane tears and swelling, typically seen in necrotic cells. It can therefore be concluded from these results that Sibutramine alters the membrane morphology of platelets to that typical of necrotic cells. Copyright © 2013 Wiley Periodicals, Inc.

  20. Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation.

    Science.gov (United States)

    Roy, Sashwati; Driggs, Jason; Elgharably, Haytham; Biswas, Sabyasachi; Findley, Muna; Khanna, Savita; Gnyawali, Urmila; Bergdall, Valerie K; Sen, Chandan K

    2011-11-01

    The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers. 2011 by the Wound Healing Society.

  1. FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

    Directory of Open Access Journals (Sweden)

    Ridolfi Ruggero

    2010-06-01

    Full Text Available Abstract Background Antigen processing by dendritic cells (DC exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials are similarly processed by these cells has not yet been resolved. Methods In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II in mature dendritic cells (mDC from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET measurements, which effectively extends the application of fluorescence microscopy to the molecular level ( Results We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.

  2. Vaccination with OK-432 followed by TC-1 tumor lysate leads to significant antitumor effects.

    Science.gov (United States)

    Chen, I-Ju; Yen, Chih-Feng; Lin, Kun-Ju; Lee, Chyi-Long; Soong, Yung-Kuei; Lai, Chyong-Huey; Lin, Cheng-Tao

    2011-07-01

    Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancer. TC-1 cell is a cell line with high expression of E7 antigen of HPV type 16 and its cell lysate has been demonstrated as an ideal inducer of E7-specific, antitumor immunity. OK-432 (Picibanil), a penicillin-killed Streptococcus pyogenes, has been reported with potent immunomodulation properties in cancer treatment by stimulating the maturation of dendritic cells (DCs) and secretion of Th-1 type cytokines. The current study demonstrated that a protocol to immunize the C57BL/6 mice with OK-432 followed by treatment with TC-1 lysate can generate markedly increased immune responses of E7-specific CD4(+) T cells and a moderate increase of natural killer (NK) cell, as well as a satisfactorily protective and therapeutic antitumor effect by triggering the DCs to prime T cells. Depletion of lymphocyte subset in vivo suggested that the antitumor effects could be dominantly executed by CD8+ T cells and followed by NK cells, and both of these reactions were induced by the generation of robust E7-specific CD4(+) T helper cell response. These findings warrant OK-432 combination with tumor-lysate as an effective and safe vaccine in future clinical application of cervical cancer.

  3. Conservation and viability of refrigerated platelet-rich plasma from New Zealand rabbits

    OpenAIRE

    Lucas Vilela Perroni Silva; Duvaldo Eurides; Marina Greco Magalhães Guerra de Andrade; Marcelo Carrijo da Costa; Vinicius Candido Ferreira; Marina de Oliveira Silva; Ednaldo Carvalho Guimarães

    2016-01-01

    Fifteen male New Zealand rabbits were used in this study, with the aim of storing their platelet-rich plasma (PRP) for 30 days at 4-6 °C to investigate its conservation and viability during this period. Thirty samples of PRP were prepared and sorted into three equal groups (G1, G2, and G3), and every three days a sample was taken out for evaluationof the number of platelets, mean platelet volume (MPV), pH of the plasma, aggregation post addition of calcium thromboplastin, and for the presence...

  4. An overview of platelet products (PRP, PRGF, PRF, etc.) in the Iranian studies.

    Science.gov (United States)

    Raeissadat, Seyed Ahmad; Babaee, Marzieh; Rayegani, Seyed Mansour; Hashemi, Zahra; Hamidieh, Amir Ali; Mojgani, Parviz; Fouladi Vanda, Hossein

    2017-11-01

    The aim of the study was to carry out a review of published studies on various platelet products in Iranian studies. Electronic databases were searched for relevant articles. Two review authors independently extracted data via a tested extraction sheet, and disagreements were resolved by a meeting with a third review author. Bone disorders (25%), wound and fistula (16%), dental and gingival disorders (14%) and osteoarthritis (11%) have more relative frequency based on different fields. The necessity of pursuing standard protocols in the preparation of platelet products, stating the precise content of platelets and growth factors, and long-term follow-up of study subjects were the most important points in Iranian studies.

  5. Extended Storage of Pathogen-Reduced Platelet Concentrates (PRECON)

    Science.gov (United States)

    2017-12-01

    transfusion. Our project proposes to determine the efficacy of using a pathogen inactivation technique (Mirasol) coupled with a platelet additive solution (PAS...technology, platelet additive solution, platelet recovery and survival, platelet storage, platelet storage solution, platelets, thrombocytopenia, transfusion...Platelets Report to 2017 Military Health System Research Symposium ……………………………………………………………….. 29 Extended Storage of Pathogen-Reduced Platelet Concentrates

  6. Effect of losartan on human platelet activation.

    Science.gov (United States)

    Guerra-Cuesta, J I; Montón, M; Rodríguez-Feo, J A; Jiménez, A M; González-Fernández, F; Rico, L A; García, R; Gómez, J; Farré, J; Casado, S; López-Farré, A

    1999-03-01

    Previous studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. Platelets were obtained from 15 healthy men, aged 26-40 years. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by the thromboxane A2 analog U46619 (5 x 10(-6) mol/l) or ADP (10(-5) mol/l). U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-dependent manner. Only a high dose of EXP 3174 (5 x 10(-5) mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I converting inhibitor, failed to modify U46619-induced platelet aggregation. Furthermore, the binding of [3H]-U46619 to platelets was competitively inhibited by losartan, whereas only a high dose of EXP 3174 reduced the binding of [3H]-U46619. Captopril failed to modify the binding of [3H]-U46619 to platelets. Losartan also reduced the platelet activation induced by ADP (10(-5) mol/l), a platelet agonist partially dependent on thromboxane A2. In addition, when thromboxane A2 generation was blocked by aspirin, ADP-induced platelet aggregation was inhibited to a similar degree to the inhibition induced by losartan. Exogenous angiotensin II did not elicit any modification of either U46619- or ADP-stimulated platelet aggregation. Losartan decreased platelet aggregation by a thromboxane A2-dependent mechanism. EXP 3174 was less potent than losartan in reducing thromboxane A2-dependent platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced thromboxane A2-dependent platelet activation independently of its effect on angiotensin II.

  7. Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage

    DEFF Research Database (Denmark)

    Sjövall, Fredrik; Ehinger, Johannes K H; Marelsson, Sigurður E

    2013-01-01

    Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source...... of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further...

  8. (Platelet aggregation in workers chronically exposed to carbon disulfide and subjected to prophylactic treatment with Cynarex)

    Energy Technology Data Exchange (ETDEWEB)

    Woyke, M.; Cwajda, H.; Wojcicki, J.; Kosmider, K.

    1981-01-01

    A group of men producing artificial fibres were administered for 2 years an artichoke extract as a preparation called Cynarex, produced by the Herbs Plant, Herbapol'' in Wroclaw. The spontaneous and ADP-induced platelets aggregation was examined by the method of Holdrinet et al. The platelets ability to aggregate, whether spontaneously or by induction was found to be statistically significantly reduced. The spontaneous aggregation after two years of Cynarex administration was reduced on average by 51%.

  9. Description of a double centrifugation tube method for concentrating canine platelets

    OpenAIRE

    Perazzi, Anna; Busetto, Roberto; Martinello, Tiziana; Drigo, Michele; Pasotto, Daniela; Cian, Francesco; Patruno, Marco; Iacopetti, Ilaria

    2013-01-01

    Background To evaluate the efficiency of platelet-rich plasma preparations by means of a double centrifugation tube method to obtain platelet-rich canine plasma at a concentration at least 4 times higher than the baseline value and a concentration of white blood cells not exceeding twice the reference range. A complete blood count was carried out for each sample and each concentrate. Whole blood samples were collected from 12 clinically healthy dogs (consenting blood donors). Blood was proces...

  10. Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

    DEFF Research Database (Denmark)

    Pleines, Irina; Eckly, Anita; Elvers, Margitta

    2010-01-01

    formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form...

  11. Encapsulation of Clay Platelets inside Latex Particles

    NARCIS (Netherlands)

    Voorn, D.J.; Ming, W.; Herk, van A.M.; Fernando, R.H.; Sung, Li-Piin

    2009-01-01

    We present our recent attempts in encapsulating clay platelets inside latex particles by emulsion polymerization. Face modification of clay platelets by cationic exchange has been shown to be insufficient for clay encapsulation, leading to armored latex particles. Successful encapsulation of

  12. Prolonged platelet preservation by transient metabolic suppression

    NARCIS (Netherlands)

    Badlou, Bahram Alamdary

    2006-01-01

    Introduction: Different clinical studies have shown that transfusion of stored platelets results in better haemostasis in patients with thrombocytopenia with and without a platelet function defect. Objectives: Current preservation procedures aim to optimally preserve the metabolic status of

  13. The nature of interactions between tissue-type plasminogen activator and platelets

    International Nuclear Information System (INIS)

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E.

    1990-01-01

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves

  14. Platelet-rich plasma, the ultimate secret for youthful skin elixir and hair growth triggering.

    Science.gov (United States)

    Elghblawi, Ebtisam

    2017-09-08

    dermatology. A literature review was conducted by searching through PubMed, Biomedical Library database, Google Scholar, and Research Gate for the terms PRP, platelet-rich plasma, platelet-rich fibrin matrix, platelet preparations, platelet application therapy, platelet growth factors, platelet facial, platelet facial rejuvenation, platelet hairs, and platelet wound healing, from inception till 2017, and they were combined using Boolean operators. All those retrieved articles in English language were looked at and explored thoroughly. © 2017 Wiley Periodicals, Inc.

  15. Toward the Relevance of Platelet Subpopulations for Transfusion Medicine

    Directory of Open Access Journals (Sweden)

    Stefan Handtke

    2018-02-01

    Full Text Available Circulating platelets consist of subpopulations with different age, maturation state and size. In this review, we address the association between platelet size and platelet function and summarize the current knowledge on platelet subpopulations including reticulated platelets, procoagulant platelets and platelets exposing signals to mediate their clearance. Thereby, we emphasize the impact of platelet turnover as an important condition for platelet production in vivo. Understanding of the features that characterize platelet subpopulations is very relevant for the methods of platelet concentrate production, which may enrich or deplete particular platelet subpopulations. Moreover, the concept of platelet size being associated with platelet function may be attractive for transfusion medicine as it holds the perspective to separate platelet subpopulations with specific functional capabilities.

  16. Potential fluid mechanic pathways of platelet activation

    OpenAIRE

    Shadden, Shawn C.; Hendabadi, Sahar

    2012-01-01

    Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here we develop a framework for computing a hemodynamic-based activation ...

  17. Platelet activation in outpatients undergoing esophagogastroduodenoscopy

    International Nuclear Information System (INIS)

    Sagripanti, A.; Polloni, A.; Materazzi, F.; Ferdeghini, M.; Pinori, E.; Bianchi, R.

    1989-01-01

    To evaluate the influence of emotional stress on platelet function mesured by radioimmunoassay in plasma two platelet factor 4, in a series of outpatients undergoing esophagogastroduodenoscopy for upper digestive complaints has been measured. The plasma levels of β-thromboglobulin and platelet factor 4, determined just before the instrumental examination, were significantly more elevated as compared to basal values, checked a week later. These results provide evidence of enhanced in vivo platelet release reaction during emotional stress

  18. Further studies on the relationship between platelet buoyant density and platelet age

    International Nuclear Information System (INIS)

    Boneu, B.; Vigoni, F.; Boneu, A.; Caranobe, C.; Sie, P.

    1982-01-01

    The relationship between platelet buoyant density and platelet age was investigated in eight human subjects submitted to an autologous chromium labeled platelet survival study. Platelets were isolated after isopycnic centrifugation using eight discontinuous isoosmotic stractan gradients (five subjects), or various continuous and linear isoosmolar gradients (three subjects). A paradoxical radioactivity enrichment of the dense platelets and a premature loss of radioactivity in the light platelets were observed. These results are explained by a shift of the radioactivity distribution curve toward higher densities during the 3-4 days after platelet injection, while the standard deviation of the distribution was conserved throughout the platelet life span. These results suggest that young platelets are heterogeneous and slightly less dense than the total platelet population

  19. Selection of donor platelets for alloimmunized patients using a platelet-associated IgG assay

    International Nuclear Information System (INIS)

    Myers, T.J.; Kim, B.K.; Steiner, M.; Baldini, M.G.

    1981-01-01

    A quantitative immunofluorescence platelet-associated immunoglobulin-G (PA-IgG) assay was used to detect alloimmunity to platelets in 8/12 multitransfused patients and to perform platelet crossmatching in the 8 alloimmunized patients. The correct separation of multitransfused patients into alloimmune and nonalloimmune groups was substantiated with chromium-51-labeled platelet survival studies. For 5 alloimmunized patients, compatible and incompatible donor platelets were demonstrated by PA-IgG crossmatching and were confirmed by platelet survival studies. With the other 3 alloimmunized patients, only Pa-IgG incompatible donor platelets were found. Survival studies with 5 of these incompatible donor platelets showed markedly reduced survival times on 4 occasions. Pa-IgG compatible donor platelets survived 3.5 to 8.7 days, while Pa-IgG incompatible platelets showed survival times of 0.1 to 2.4 days

  20. Platelet function testing in pediatric patients

    DEFF Research Database (Denmark)

    Hvas, Anne-Mette; Favaloro, Emmanuel J

    2017-01-01

    Introduction: Platelets play a key role in primary hemostasis and are also intricately linked to secondary hemostasis. Investigation of platelet function in children, especially in neonates, is seriously challenged by the volumes required to perform the majority of platelet function tests and due...

  1. Platelet counting using the Coulter electronic counter.

    Science.gov (United States)

    Eggleton, M J; Sharp, A A

    1963-03-01

    A method for counting platelets in dilutions of platelet-rich plasm using the Coulter electronic counter is described.(1) The results obtained show that such platelet counts are at least as accurate as the best methods of visual counting. The various technical difficulties encountered are discussed.

  2. Platelet kinetics: the state of the art

    International Nuclear Information System (INIS)

    Heyns, A. duP

    1984-01-01

    In this paper an overview of the state of the art of platelet kinetics 1982 is presented. The subjects considered include a discussion of the advantages and disadvantages of some of the many radionuclide platelet labels, viz 51 Cr, 111 In, focussing briefly on models for analysis of platelets survival. (Auth.)

  3. Evaluating Platelet-Rich Therapy for Facial Aesthetics and Alopecia: A Critical Review of the Literature.

    Science.gov (United States)

    Motosk O, Catherine C; Khouri, Kimberly S; Poudrier, Grace; Sinno, Sammy; Hazen, Alexes

    2018-05-01

    Despite the growing popularity of platelet-rich plasma, existing evidence supporting its efficacy remains controversial due to the lack of large-scale studies and standardized protocols for preparation and application. This article reviews its use in facial rejuvenation, fat grafting, acne scarring, and androgenic alopecia. Emphasis is placed on comparing methods of platelet-rich plasma preparation and application across studies. A systematic review was performed for articles published between 2006 and 2015. All clinical studies and case reports that addressed platelet-rich plasma alone and/or in combination with fat grafting for facial rejuvenation, acne scarring, or androgenic alopecia were included. Of the 22 articles included in the analysis, seven studies used platelet-rich plasma alone for facial rejuvenation, seven in combination with fat grafting, two for treatment of acne scarring, and six for treatment of androgenic alopecia. Individual study procedures, means of evaluation, and significant results are summarized. Although the majority of studies in this review report positive results, significant variation exists in preparation protocols and in the number and frequency of clinical treatments. The majority of studies report positive results for all indications evaluated in this review, but the procedure is limited by the lack of a standardized method for preparation and application of platelet-rich plasma. The extent to which significant variability in platelet-rich plasma preparation and/or application methods may affect clinical outcomes is not completely clear. In the interim, we present a consolidation of platelet-rich plasma treatment techniques and outcomes currently in use to help guide physicians in their clinical practice.

  4. Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus

    Directory of Open Access Journals (Sweden)

    Josiane M. Pazzini

    2016-01-01

    Full Text Available Abstract: Platelet-rich plasma (PRP is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood

  5. Effect of ionizing radiation on platelet function in vitro

    International Nuclear Information System (INIS)

    Kalovidouris, A.E.; Papayannis, A.G.

    1981-01-01

    The effect of ionizing radiation on platelet function was investigated in vitro. Platelet-rich plasma (300x10 9 /l) was irradiated with doses of 1, 4, 10, 20 and 50 Gy. Platelet function tests were performed on both irradiated and control (non-irradiated) platelet samples. The platelet function tests were (1) platelet aggregation by ADP (1, 2, 4 μmol final concentration), adrenaline and collagen, (2) ADP-release from platelets, (3) clot retraction and (4) platelet factor-3 availability. It was found that roentgen irradiation of platelets in vitro did not affect these platelet function tests. (Auth.)

  6. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

    Directory of Open Access Journals (Sweden)

    Iris Bosschem

    Full Text Available Helicobacter suis (H. suis is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin, administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC/lysate and intranasal immunization with Cholera toxin (CT/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

  7. Platelet count and platelet indices in women with preeclampsia.

    Science.gov (United States)

    AlSheeha, Muneera A; Alaboudi, Rafi S; Alghasham, Mohammad A; Iqbal, Javed; Adam, Ishag

    2016-01-01

    Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia. To compare platelet indices, namely platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), and PC to MPV ratio in women with preeclampsia compared with healthy controls. Qassim Hospital, Kingdom of Saudi Arabia. A case-control study. Sixty preeclamptic women were the cases and an equal number of healthy pregnant women were the controls. There was no significant difference in age, parity, and body mass index between the study groups. Sixteen and 44 of the cases were severe and mild preeclampsia, respectively. There was no significant difference in PDW and MPV between the preeclamptic and control women. Both PC and PC to MPV ratios were significantly lower in the women with preeclampsia compared with the controls. There was no significant difference in the PC, PDW, MPV, and PC to MPV ratio when women with mild and severe preeclampsia were compared. Using receiver operating characteristic (ROC) curves, the PC cutoff was 248.0×10 3 /µL for diagnosis of pre-eclampsia ( P =0.019; the area under the ROC curve was 62.4%). Binary regression suggests that women with PC preeclampsia (odds ratio =2.2, 95% confidence interval =1.08-4.6, P =0.03). The PC/MPV cutoff was 31.2 for diagnosis of preeclampsia ( P =0.035, the area under the ROC curve was 62.2%). PC preeclampsia.

  8. Inhibition of Platelet Aggregation by the Leaf Extract of Carica papaya During Dengue Infection: An In Vitro Study.

    Science.gov (United States)

    Chinnappan, Shobia; Ramachandrappa, Vijayakumar Shettikothanuru; Tamilarasu, Kadhiravan; Krishnan, Uma Maheswari; Pillai, Agiesh Kumar Balakrishna; Rajendiran, Soundravally

    2016-04-01

    Dengue cases were reported to undergo platelet activation and thrombocytopenia by a poorly understood mechanism. Recent studies suggested that Carica papaya leaf extract could recover the platelet count in dengue cases. However, no studies have attempted to unravel the mechanism of the plant extract in platelet recovery. Since there are no available drugs to treat dengue and considering the significance of C. papaya in dengue treatment, the current study aimed to evaluate two research questions: First one is to study if the C. papaya leaf extract exerts its action directly on platelets and second one is to understand if the extract can specifically inhibit the platelet aggregation during dengue viral infection. Sixty subjects with dengue positive and 60 healthy subjects were recruited in the study. Platelet-rich plasma (PRP) and platelet-poor plasma were prepared from both the dengue-infected and healthy control blood samples. Effect of the leaf extract obtained from C. papaya leaves was assessed on plasma obtained as well as platelets collected from both healthy and dengue-infected individuals. Platelet aggregation was significantly reduced when leaf extract preincubated with dengue plasma was added into control PRP, whereas no change in aggregation when leaf extract incubated-control plasma was added into control PRP. Upon direct addition of C. papaya leaf extract, both dengue PRP and control PRP showed a significant reduction in platelet aggregation. Within the dengue group, PRP from severe and nonsevere cases showed a significant decrease in aggregation without any difference between them. From the study, it is evident that C. papaya leaf extract can directly act on platelet. The present study, the first of its kind, found that the leaf extract possesses a dengue-specific neutralizing effect on dengue viral-infected plasma that may exert a protective role on platelets.

  9. [125I] radioiodinated metaraminol: A new platelet-specific labeling agent

    International Nuclear Information System (INIS)

    Ohmomo, Y.; Yokoyama, A.; Kawai, K.; Arano, Y.; Horiuchi, K.; Saji, H.; Torizuka, K.

    1985-01-01

    In our search for a platelet-specific labeling agent, metaraminol (MA), a low-toxic pharmaceutical for the treatment of hypotension and cardiogenic shock, attracted our attention. Its active incorporation and accumulation by platelets have been recognized. At first, the preparation of 125 I radioiodinated metaraminol ( 125 I-MA) was carried out using the chloramine-T method. Then, upon the harvest of platelets as platelet-rich plasma (PRP), their labeling with this new radiopharmaceutical was easily performed by incubation for 10 min at 37 0 C. The cell-labeling efficiency was dependent on cell density, reaching 63.0%+-3.1% at 2.4x10 9 cells/ml. The specific incorporation of 125 I-MA by an active transport system similar to that of 5-hydroxytryptamine (5-HT) as well as by passive diffusion was demonstrated. In vitro studies, the unaltered state of 125 I-MA-labeled platelets with their cellular functions fully retained was estimated. In vivo studies carried out in rabbits with induced thrombi in the femoral artery showed a rather rapid disappearance of the radioactivity from circulating blood, reaching a high thrombus-to-blood activity ratio of 19.8+-4.3 within 30 min of the administration of 125 I-MA-labeled autologous platelets. Thus, with the potential availability of 123 I, 123 I-MA-labeled platelets appear to be a promising agent for thrombus imaging using single-emission computed tomography (CT) studies. (orig.)

  10. Role of platelets in maintenance of pulmonary vascular permeability to protein

    International Nuclear Information System (INIS)

    Lo, S.K.; Burhop, K.E.; Kaplan, J.E.; Malik, A.B.

    1988-01-01

    The authors examined the role of platelets in maintenance of pulmonary vascular integrity by inducing thrombocytopenia in sheep using antiplatelet serum (APS). A causal relationship between thrombocytopenia and increase in pulmonary vascular permeability was established by platelet repletion using platelet-rich plasma (PRP). Sheep were chronically instrumented and lung lymph fistulas prepared to monitor pulmonary lymph flow (Q lym ). A balloon catheter was positioned in the left atrium to assess pulmonary vascular permeability to protein after raising the left atrial pressure (P la ). Thrombocytopenia was maintained for 3 days by daily intramuscular APS injections. In studies using cultured bovine pulmonary artery endothelial monolayers, transendothelia permeability of 125 I-labeled albumin was reduced 50 and 95%, respectively, when 2.5 x 10 7 or 5 x 10 7 platelets were added onto endothelial monolayers. However, addition of 5 x 10 6 platelets or 5 x 10 7 red blood cells did not reduce endothelial monolayer albumin permeability. Results indicate that platelets are required for the maintenance of pulmonary vascular permeability. Reduction in permeability appears to involve an interaction of platelets with the endothelium

  11. Effect of hyperbaric oxygen therapy combined with autologous platelet concentrate applied in rabbit fibula fraction healing

    Directory of Open Access Journals (Sweden)

    Paulo Cesar Fagundes Neves

    2013-09-01

    Full Text Available OBJECTIVES: The purpose is to study the effects of hyperbaric oxygen therapy and autologous platelet concentrates in healing the fibula bone of rabbits after induced fractures. METHODS: A total of 128 male New Zealand albino rabbits, between 6-8 months old, were subjected to a total osteotomy of the proximal portion of the right fibula. After surgery, the animals were divided into four groups (n = 32 each: control group, in which animals were subjected to osteotomy; autologous platelet concentrate group, in which animals were subjected to osteotomy and autologous platelet concentrate applied at the fracture site; hyperbaric oxygen group, in which animals were subjected to osteotomy and 9 consecutive daily hyperbaric oxygen therapy sessions; and autologous platelet concentrate and hyperbaric oxygen group, in which animals were subjected to osteotomy, autologous platelet concentrate applied at the fracture site, and 9 consecutive daily hyperbaric oxygen therapy sessions. Each group was divided into 4 subgroups according to a pre-determined euthanasia time points: 2, 4, 6, and 8 weeks postoperative. After euthanasia at a specific time point, the fibula containing the osseous callus was prepared histologically and stained with hematoxylin and eosin or picrosirius red. RESULTS: Autologous platelet concentrates and hyperbaric oxygen therapy, applied together or separately, increased the rate of bone healing compared with the control group. CONCLUSION: Hyperbaric oxygen therapy and autologous platelet concentrate combined increased the rate of bone healing in this experimental model.

  12. Large-scale preparation of plasmid DNA.

    Science.gov (United States)

    Heilig, J S; Elbing, K L; Brent, R

    2001-05-01

    Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

  13. Evidence that platelet buoyant density, but not size, correlates with platelet age in man

    International Nuclear Information System (INIS)

    Mezzano, D.; Hwang, K.; Catalano, P.; Aster, R.H.

    1981-01-01

    Following infusion of 51Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radio-activity of LD platelets declined rapidly post-infusion (T1/2 . 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3--4-day period and gradually declined for 6--7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P less than 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33) days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P less than 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets . 7.57 mu3, LD platelets . 6.87 mu3, 0.05 less than P less than 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions

  14. Methods for evaluation of platelet function.

    Science.gov (United States)

    Lindahl, Tomas L; Ramström, Sofia

    2009-10-01

    There are a multitude of platelet function tests available, reflecting the complex nature of the platelet in haemostasis. No simple single test will ever cover all aspects of platelet function. Some tests focus on the aggregation of platelets, for example aggregometry, other on the swelling in response to hypotonic solutions, i.e. the well-known hypotonic shock response, or adhesion or coagulation and clot retraction, for example thromboelastography. In general there is a lack of clinical studies showing a predictive value of analysis of platelet concentrates.

  15. Defining Platelet Function During Polytrauma

    Science.gov (United States)

    2014-04-01

    scaffold of the clot after its thrombin-induced polymerization to fibrin fibers. Formation of a stiff fibrin fiber network and its contraction by platelet...In another study (PROTEC T) Refused Participat ion (Yes=Y, No=N, W=Unqua lified , Refused further draws (agreed to keep previous data

  16. Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

    OpenAIRE

    Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

    1994-01-01

    We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The...

  17. Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates

    Directory of Open Access Journals (Sweden)

    Yinliang Yang

    2013-10-01

    Full Text Available Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions.

  18. Anti-platelet activity of water dispersible curcuminoids in rat platelets.

    Science.gov (United States)

    Maheswaraiah, Anikisetty; Rao, Lingamallu Jaganmohan; Naidu, Kamatham Akhilender

    2015-03-01

    Curcuminoids are active principle of turmeric with plethora of health beneficial properties. In this study, we have evaluated for the first time the effect of water dispersible curcuminoids on rat platelet aggregation. Curcuminoids (10-30 µg/mL) significantly inhibited platelet aggregation induced by agonists viz., collagen, ADP and arachidonic acid. Curcuminoids were found to be two-fold more potent than curcumin in inhibiting platelet aggregation. Intracellular curcuminoid concentration was relatively higher than curcumin in rat platelets. Curcuminoids significantly attenuated thromboxane A2 , serotonin levels in rat platelets which play an important role in platelet aggregation. Curcuminoid treatment increased nitric oxide (NO) levels in platelets treated with agonists. Curcuminoids inhibited free radicals such as superoxide anion released from activated platelets, which ultimately inhibits platelet aggregation. Further, curcuminoids inhibited 12-lipoxygenase activity and formation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) in activated rat platelets which regulates platelet aggregation. The results suggest that curcuminoids have remarkable anti-platelet activity by modulating multiple mechanisms involved in platelet aggregation. Thus curcuminoids may have a therapeutic potential to prevent platelet activation related disorders. Copyright © 2015 John Wiley & Sons, Ltd.

  19. Determination of Immunogenic Relevant Antigens in the Excretory-Secretory (ES Products and the Lysates of Ascaridia galli Larvae

    Directory of Open Access Journals (Sweden)

    S Saffi

    2008-04-01

    Full Text Available Background: Ascaridia galli, the largest nematode of small intestine of birds, especially the native poultry, may give rise to serious illness, pathological defects and economical losses even in modern poultry production systems. Although various measures have been undertaken to vaccinate poultry against A.galli, no satisfactory results were obtained so far. However, there is no report on the efficacy of excretory-secretory (ES proteins of A.galli larvae in immunization of poultry. Thus, the aim of the present research project was based on the use of the ES products of the larvae, in order to find the protective anti­gens.Methods: Five hundred native poultry were autopsied and adult A.galli was removed form their intestines. The eggs were harvested form the uterus of female worms and cultured at 25 ˚C in water containing 0.1 N sulphuric acid for almost a fort­night. The larvae were then freed mechanically and kept in Earl's salt solution for a few days. The supernatant solution of alive larvae containing the ES products of the larvae, as well as the sonicated alive and dead larvae, was analyzed by SDS-PAGE.Result: Many protein fractions of 15 kDa up to 200 kDa were demonstrated in lysate of these larvae. Using the serum of a hen, infected with a high numbers of A.galli, an immunogenic antigen was identified between 55 kDa to 72 kDa by Western blotting procedure.Conclusion: Finding the protein band between 55 and 72 kDa can be promising for preparation of vaccine, though more investigations are needed to prove the protective ability of this antigen.

  20. Evaluation of two platelet-rich plasma processing methods and two platelet-activation techniques for use in llamas and alpacas.

    Science.gov (United States)

    Semevolos, Stacy A; Youngblood, Cori D; Grissom, Stephanie K; Gorman, M Elena; Larson, Maureen K

    2016-11-01

    OBJECTIVE To evaluate 2 processing methods (commercial kit vs conical tube centrifugation) for preparing platelet rich plasma (PRP) for use in llamas and alpacas. SAMPLES Blood samples (30 mL each) aseptically collected from 6 healthy llamas and 6 healthy alpacas. PROCEDURES PRP was prepared from blood samples by use of a commercial kit and by double-step conical tube centrifugation. A CBC was performed for blood and PRP samples. Platelets in PRP samples were activated by means of a freeze-thaw method with or without 23mM CaCl 2 , and concentrations of platelet-derived growth factor-BB and transforming growth factor-β 1 were measured. Values were compared between processing methods and camelid species. RESULTS Blood CBC values for llamas and alpacas were similar. The commercial kit yielded a significantly greater degree of platelet enrichment (mean increase, 8.5 fold vs 2.8 fold) and WBC enrichment (mean increase, 3.7 fold vs 1.9 fold) than did conical tube centrifugation. Llamas had a significantly greater degree of platelet enrichment than alpacas by either processing method. No difference in WBC enrichment was identified between species. Concentrations of both growth factors were significantly greater in PRP samples obtained by use of the commercial kit versus those obtained by conical tube centrifugation. CONCLUSIONS AND CLINICAL RELEVANCE For blood samples from camelids, the commercial kit yielded a PRP product with a higher platelet and WBC concentration than achieved by conical tube centrifugation. Optimal PRP platelet and WBC concentrations for various applications need to be determined for llamas and alpacas.

  1. [The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

    Science.gov (United States)

    Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

    2014-08-01

    The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

  2. Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide microarray.

    Science.gov (United States)

    Sharif, Suhela; Shi, Jie; Bourakba, Mostafa; Ruijtenbeek, Rob; Pieters, Roland J

    2017-09-01

    O-GlcNAcylation is a post-translational modification resulting from the addition of an N-acetylglucosamine moiety to the hydroxyl groups of serine and threonine residues of nuclear and cytoplasmic proteins. In addition, O-GlcNAcylated proteins can be phosphorylated, which suggests the possibility for crosstalk between O-GlcNAcylation and phosphorylation. Dysregulation of O-GlcNAcylation affects cell signaling, transcriptional regulation, cell cycle control and can e.g. lead to tumorigenesis and tumor metastasis. There is a strong demand for efficient analytical techniques to better detect and investigate this abundant modification and its role in cancer. Herein we demonstrated the utility of an O-GlcNAcylated peptide array to examine O-GlcNAcase (OGA) activity and substrate specificity of both purified protein as well cell lysates of different cancer cell lines. Using this microarray, we clearly observed OGA activity and also inhibition thereof by OGA inhibitor thiamet G. Interestingly, different levels of OGA activity were observed of lysates derived from different cancer cell lines. This suggests that the tool may be useful in cancer research and biomarker development. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

    NARCIS (Netherlands)

    Pietersz, R. N.; Loos, J. A.; Reesink, H. W.

    1987-01-01

    High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in

  4. Thrombokinetics with In-111-oxine labelled platelets

    International Nuclear Information System (INIS)

    Uchida, Tatsumi; Yui, Tokuo; Muroi, Shuichi; Matsuda, Shin; Kariyone, Shigeo

    1982-01-01

    Indium-111-oxine has been employed as a redioactive platelet label for thrombosis imaging and thrombokinetic studies in man. To evaluate it's suitability for platelet survival and turnover, thrombokinetic studies were carried out in hematological normal subjects, in patients with idiopathic thrombocytopenic purpura (ITP) and chronic congestive splenomegaly. For In-111-oxine labelled platelets, platelets were collected by differential centrifugation from 44 ml of whole blood drawn into 6 ml of acid citrate dextrose solution. Platelet suspension was incubated with In-111-oxine, which was extracted before use by the method of Thakur and co-workers. The survival, recovery and turnover of In-111-labeled platelets were 8.6 +- 0.5 days, 63.0 +- 5.4% and 3.9 +- 0.3 x 10 4 / μl/day, respectively, which were similar with those of Cr-51 method. Platelet disappearance curves labelled with In-111 and Cr-51 simultaneously were similar in one case. In patients with ITP, platelet survival shortened in the same degree with Cr-51 method. The two simultaneous labeling studies between In-111 and Cr-51 showed no differences. In the patients with congestive splenomegaly, the same results were obtained. Thrombokinetic studies with In-111-oxine labelled platelets offer the advantages of reduced blood requirements, and the ability to perform external imaging of platelet distribution. (author)

  5. Influence of Oxidative Stress on Stored Platelets

    Directory of Open Access Journals (Sweden)

    K. Manasa

    2016-01-01

    Full Text Available Platelet storage and its availability for transfusion are limited to 5-6 days. Oxidative stress (OS is one of the causes for reduced efficacy and shelf-life of platelets. The studies on platelet storage have focused on improving the storage conditions by altering platelet storage solutions, temperature, and materials. Nevertheless, the role of OS on platelet survival during storage is still unclear. Hence, this study was conducted to investigate the influence of storage on platelets. Platelets were stored for 12 days at 22°C. OS markers such as aggregation, superoxides, reactive oxygen species, glucose, pH, lipid peroxidation, protein oxidation, and antioxidant enzymes were assessed. OS increased during storage as indicated by increments in aggregation, superoxides, pH, conjugate dienes, and superoxide dismutase and decrements in glucose and catalase. Thus, platelets could endure OS till 6 days during storage, due to the antioxidant defense system. An evident increase in OS was observed from day 8 of storage, which can diminish the platelet efficacy. The present study provides an insight into the gradual changes occurring during platelet storage. This lays the foundation towards new possibilities of employing various antioxidants as additives in storage solutions.

  6. Detection of microbial contamination in platelets

    Science.gov (United States)

    Berg, Tracy L.; Leparc, German; Huffman, Debra E.; Gennaccaro, Angela L.; Garcia-Lopez, Alicia; Klungness, Greta; Stephans, Christie; Garcia-Rubio, Luis H.

    2005-03-01

    In the United States, approximately 100 patients develop fatal sepsis associated with platelet transfusions every year. Current culture methods take 24-48 hours to acquire results, which in turn decrease the shelf life of platelets. Many of the microorganisms that contaminate platelets can replicate easily at room temperature, which is the necessary storage temperature to keep platelets functional. Therefore, there is a need for in-situ quality control assessment of the platelet quality. For this purpose, a real time spectrophotometric technique has been developed. The Spectral Acquisition Processing Detection (SAPD) method, comprised of a UV-vis spectrophotometer and modeling algorithms, is a rapid method that can be performed prior to platelet transfusion to decrease the risk of bacterial infection to patients. The SAPD method has been used to determine changes in cell suspensions, based on size, shape, chemical composition and internal structure. Changes in these cell characteristics can in turn be used to determine microbial contamination, platelet aging and other physiologic changes. Detection limits of this method for platelet suspensions seeded with bacterial contaminants were identified to be less than 100 cfu/ml of sample. Bacterial counts below 1000 cfu/ml are not considered clinically significant. The SAPD method can provide real-time identification of bacterial contamination of platelets affording patients an increased level of safety without causing undue strain on laboratory budgets or personnel while increasing the time frame that platelets can be used by dramatically shortening contaminant detection time.

  7. The role of platelets during reproduction.

    Science.gov (United States)

    Isermann, Berend; Nawroth, Peter P

    2006-01-01

    The availability of mice with defined defects within the hemostatic system enabled researchers to identify a role the coagulation system for embryonic and placental development. However, the role of platelets during development has only recently been experimentally addressed, giving some insight into potential functions of platelets during development. Thus, a quantitative embryonic platelet defect (severe thrombopenia secondary to NF-E2 deficiency) is associated with an embryonic growth retardation and reduced vascularisation of the placenta. Maternal platelet deficiency is associated with placental hemorrhage, which, however, does not impair embryonic or maternal survival. In vitro studies established that platelets or platelet conditioned medium regulate the invasive properties of human extravillous trophoblast cells and induce a phenotypical switch of trophoblast cells. These data imply that platelets are of relevance during placentation. Conversely, platelets and the formation of platelet-fibrin aggregates are dispensable for the development of the embryo proper, establishing that the lethal phenotypes observed in some embryo slacking coagulation regulators does not result from an inability to form platelet-fibrin aggregates, but likely reflects altered protease dependent signaling during vascular development.

  8. Platelet destruction in autoimmune thrombocytopenic purpura: kinetics and clearance of indium-111-labeled autologous platelets

    International Nuclear Information System (INIS)

    Stratton, J.R.; Ballem, P.J.; Gernsheimer, T.; Cerqueira, M.; Slichter, S.J.

    1989-01-01

    Using autologous 111 In-labeled platelets, platelet kinetics and the sites of platelet destruction were assessed in 16 normal subjects (13 with and three without spleens), in 17 studies of patients with primary autoimmune thrombocytopenic purpura (AITP), in six studies of patients with secondary AITP, in ten studies of patients with AITP following splenectomy, and in five thrombocytopenic patients with myelodysplastic syndromes. In normal subjects, the spleen accounted for 24 +/- 4% of platelet destruction and the liver for 15 +/- 2%. Untreated patients with primary AITP had increased splenic destruction (40 +/- 14%, p less than 0.001) but not hepatic destruction (13 +/- 5%). Compared with untreated patients, prednisone treated patients did not have significantly different spleen and liver platelet sequestration. Patients with secondary AITP had similar platelet counts, platelet survivals, and increases in splenic destruction of platelets as did patients with primary AITP. In contrast, patients with myelodysplastic syndromes had a normal pattern of platelet destruction. In AITP patients following splenectomy, the five nonresponders all had a marked increase (greater than 45%) in liver destruction compared to five responders (all less than 40%). Among all patients with primary or secondary AITP, there was an inverse relationship between the percent of platelets destroyed in the liver plus spleen and both the platelet count (r = 0.75, p less than 0.001) and the platelet survival (r = 0.86, p less than 0.001). In a stepwise multiple linear regression analysis, total liver plus spleen platelet destruction, the platelet survival and the platelet turnover were all significant independent predictors of the platelet count. Thus platelet destruction is shifted to the spleen in primary and secondary AITP. Failure of splenectomy is associated with a marked elevation in liver destruction

  9. Indium-111 platelet imaging for detection of platelet deposition in abdominal aneurysms and prosthetic arterial grafts

    International Nuclear Information System (INIS)

    Ritchie, J.L.; Stratton, J.R.; Thiele, B.; Haminton, G.W.; Warrick, L.N.; Huang, T.W.; Harker, L.A.

    1981-01-01

    Thirty-four platelet imaging studies were performed in 23 patients to determine whether platelet deposition could be detected in patients with vascular aneurysms (18 patients) or in patients in whom Dacron prosthetic grafts had been placed (5 patients). In patients in whom abnormal platelet deposition was detected, the effect of administration of platelet-active drugs on platelet deposition was examined. Of the 18 patients with an aneurysm, 12 had equivocally positive studies on initial imaging and 2 had equivocally positive images. Of five patients with Dacron arterial grafts in place, four had diffuse platelet deposition in the grafts; the fifth patient had a platelet deposition only in a pseudoaneurysm. Eight patients with an abdominal aneurysm and positive or equivocally positive baseline images were restudied during platelet-active drug therapy either with aspirin plus dipyridamole (seven patients) or with sulfinpyrazone (four patients). No patient studied during treatment with aspirin plus dipyridamole had detectably decreased platelet deposition compared with baseline determinations. In contrast, two of four patients studied while receiving sulfinpyrazone showed decreased platelet deposition. Thus, platelet imaging may be of value for studying platelet physiology in vivo and for assessing platelet-active drugs and the thrombogenicity of prosthetic graft materials in human beings

  10. Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS.

    Science.gov (United States)

    Walpurgis, Katja; Kohler, Maxie; Thomas, Andreas; Wenzel, Folker; Geyer, Hans; Schänzer, Wilhelm; Thevis, Mario

    2012-08-01

    The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Three-dimensional structure and cytokine distribution of platelet-rich fibrin.

    Science.gov (United States)

    Bai, Meng-Yi; Wang, Ching-Wei; Wang, Jyun-Yi; Lin, Ming-Fang; Chan, Wing P

    2017-02-01

    Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1) can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg); it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5) to low at the plasma end (p=0.19). Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

  12. Three-dimensional structure and cytokine distribution of platelet-rich fibrin

    Directory of Open Access Journals (Sweden)

    Meng-Yi Bai

    Full Text Available OBJECTIVES: Previous reports have revealed that several cytokines (including platelet-derived growth factor-BB, transforming growth factors-β1 and insulin-like growth factor-1 can enhance the rate of bone formation and synthesis of extracellular matrix in orthopaedics or periodontology. This study aimed to determine the concentration of cytokines within platelet-rich fibrin microstructures and investigate whether there are differences in the different portions of platelet-rich fibrin, which has implications for proper clinical use of platelet-rich fibrin gel. METHODS: Whole blood was obtained from six New Zealand rabbits (male, 7 to 39 weeks old, weight 2.7-4 kg; it was then centrifuged for preparation of platelet-rich fibrin gels and harvest of plasma. The resultant platelet-rich fibrin gels were used for cytokine determination, histological analyses and scanning electron microscopy. All plasmas obtained were subject to the same cytokine determination assays for the purpose of comparison. RESULTS: Cytokines platelet-derived growth factor-BB and transforming growth factor-β1 formed concentration gradients from high at the red blood cell end of the platelet-rich fibrin gel (p=1.88×10-5 to low at the plasma end (p=0.19. Insulin-like growth factor-1 concentrations were similar at the red blood cell and plasma ends. The porosities of the platelet-rich fibrin samples taken in sequence from the red blood cell end to the plasma end were 6.5% ± 4.9%, 24.8% ± 7.5%, 30.3% ± 8.5%, 41.4% ± 12.3%, and 40.3% ± 11.7%, respectively, showing a gradual decrease in the compactness of the platelet-rich fibrin network. CONCLUSION: Cytokine concentrations are positively associated with platelet-rich fibrin microstructure and portion in a rabbit model. As platelet-rich fibrin is the main entity currently used in regenerative medicine, assessing cytokine concentration and the most valuable portion of PRF gels is essential and recommended to all physicians.

  13. Standardization of a Protocol for Obtaining Platelet Rich Plasma from blood Donors; a Tool for Tissue Regeneration Procedures.

    Science.gov (United States)

    Gómez, Lina Andrea; Escobar, Magally; Peñuela, Oscar

    2015-01-01

    To develop a protocol for obtaining autologous platelet rich plasma in healthy individuals and to determine the concentration of five major growth factors before platelet activation. This protocol could be integrated into the guidelines of good clinical practice and research in regenerative medicine. Platelet rich plasma was isolated by centrifugation from 38 healthy men and 42 women ranging from 18 to 59 years old. The platelet count and quantification of growth factors were analyzed in eighty samples, stratified for age and gender of the donor. Analyses were performed using parametric the t-test or Pearson's analysis for non-parametric distribution. P platelet counts from 1.6 to 4.9 times (mean = 2.8). There was no correlation between platelet concentration and the level of the following growth factors: VEGF-D (r = 0.009, p = 0.4105), VEGF-A (r = 0.0068, p = 0.953), PDGF subunit AA (p = 0.3618; r = 0.1047), PDGF-BB (p = 0.5936; r = 0.6095). In the same way, there was no correlation between donor gender and growth factor concentrations. Only TGF-β concentration was correlated to platelet concentration (r = 0.3163, p = 0.0175). The procedure used allowed us to make preparations rich in platelets, low in leukocytes and red blood cells, and sterile. Our results showed biological variations in content of growth factors in PRP. The factors influencing these results should be further studied.

  14. Growth factor and proteinase profile of Vivostat® platelet-rich fibrin linked to tissue repair.

    Science.gov (United States)

    Agren, M S; Rasmussen, K; Pakkenberg, B; Jørgensen, B

    2014-07-01

    Autologous platelet-rich fibrin (PRF(®)) is prepared by the automatic Vivostat(®) system. Conflicting results with Vivostat PRF in acute wound healing prompted us to examine its cellular and biomolecular composition. Specifically, platelets, selected growth factors and matrix metalloproteinase (MMP)-9 were quantified using novel analytical methods. Ten healthy non-thrombocytopenic volunteers donated blood for generation of intermediate fibrin-I and final PRF. Anticoagulated whole blood and serum procured in parallel served as baseline controls. Leucocyte, erythrocyte and platelet counts in whole blood and fibrin-I were determined by automated haematology analyser. Platelet concentration in PRF was quantified manually by stereologic analysis of Giemsa-stained tissue sections, and the total content of five growth factors and MMP-9 by enzyme-linked immunosorbent assays. The number of leucocytes and erythrocytes was reduced (P platelets increased (P fibrin-I versus whole blood. PRF contained 982 ± 206 × 10(9) platelets/l representing 3·9-fold (P platelet-derived growth factor (PDGF)-AB [2·5-fold, P PDGF-BB [1·6-fold, P vascular endothelial growth factor > basic fibroblast growth factor [75-fold, P platelet enrichment and biomolecular constituents may guide clinicians in their optimal use of Vivostat PRF for tissue regenerative applications. © 2013 International Society of Blood Transfusion.

  15. Therapeutic platelet reduction: Use in postsplenectomy thrombocytosis

    Directory of Open Access Journals (Sweden)

    Gita Negi

    2015-01-01

    Full Text Available Therapeutic platelet reduction is an effective modality for the reduction of platelet count in patients with treatment of extreme thrombocytosis resulting from a variety of primary and secondary causes of thrombocytosis, which may be associated with thrombotic or hemorrhagic complications of varying degrees. These cases when symptomatic fall into the ASFA Category II indication for therapeutic platelet apheresis procedure. Here, we report a case of postsplenectomy secondary thrombocytosis presenting with extremely high platelet counts and subsequent thrombosis in the shunt and successful treatment after therapeutic platelet reduction. The case is being presented to bring forth the fact that therapeutic platelet reduction is an easy procedure that gives quick and good results and also to bring to the attention of transfusion specialists an associated but as yet unreported procedural finding.

  16. Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

    Science.gov (United States)

    Petrzik, K; Mráz, I; Kubelková, D

    2001-02-01

    The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

  17. LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

    Science.gov (United States)

    Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

    2014-11-01

    Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Comparative studies on the corrosion protection effect of DBSA-doped polyaniline prepared from in situ emulsion polymerization in the presence of hydrophilic Na+-MMT and organophilic organo-MMT clay platelets

    International Nuclear Information System (INIS)

    Chang, K.-C.; Lai, M.-C.; Peng, C.-W.; Chen, Y.-T.; Yeh, J.-M.; Lin, C.-L.; Yang, J.-C.

    2006-01-01

    A series of polyaniline (PANI)/Na + -montmorillonite (MMT) clay and PANI/organo-MMT nanocomposite materials have been successfully prepared by in situ emulsion polymerization in the presence of inorganic nanolayers of hydrophilic Na + -MMT clay or organophilic organo-MMT clay with DBSA and KPS as surfactant and initiator, respectively. The as-synthesized Na + -PCN and organo-PCN materials were characterized and compared by Fourier transformation infrared (FTIR) spectroscopy, wide-angle powder X-ray diffraction (XRD) and transmission electron microscopy (TEM). Na + -PCN materials in the form of coatings with low loading of Na + -MMT clay (e.g., 3 wt.%, CLAN3) on cold-rolled steel (CRS) were found much superior in corrosion protection over those of organo-PCN materials with same clay loading based on a series of electrochemical measurements of corrosion potential, polarization resistance, corrosion current and impedance spectroscopy in 5 wt.% aqueous NaCl electrolyte. The molecular weights of PANI extracted from PCN materials and neat PANI were determined by gel permeation chromatography (GPC) with NMP as eluant. Effects of material composition on the gas permeability, optical properties and electrical conductivity of neat PANI and a series of PCN materials, in the form of free-standing film, solution and powder-pressed pellet, were also studied by gas permeability analyzer (GPA), ultraviolet-vis spectra and four-point probe technique, respectively

  19. Heat inactivation of a norovirus surrogate in cell culture lysate, abalone meat, and abalone viscera.

    Science.gov (United States)

    Park, Shin Young; Bae, San-Cheong; Ha, Sang-Do

    2015-03-01

    The current study examined the effects of temperature and heat treatment duration on murine norovirus-1 (MNV-1) from both viral cell culture lysate (7-8 log10 PFU) and experimentally contaminated abalone meat and viscera (5-6 log10 PFU) as a model of human norovirus (NoV). MNV-1 titers in cell culture lysate, abalone meat, and abalone viscera were gradually reduced to 1.93-4.55, 1.79-3.00, and 2.26-3.26 log10 PFU/ml, respectively, after treatment at 70 °C for 1-10 min. Treatment at 85 °C for 1-5 min gradually reduced MNV-1 titers in abalone meat to 2.71-4.15 log10 PFU/ml. MNV-1 titers in abalone viscera were gradually reduced to 2.91-3.46 log10 PFU/ml after treatment at 85 °C for 1-3 min. No significant difference (P > 0.05) was found in MNV-1 titers in the abalone meat and viscera among treatment groups (70 °C for 5 min, 70 °C for 3 min, and 85 °C for 1 min). Complete inactivation of MNV-1 in cell culture lysate was determined at 85 °C for ≥1 min and 100 °C for ≥0.5 min. Complete inactivation of MNV-1 in abalone was determined at 100 °C for ≥0.5 min for meat, and 85 °C for 5 min and 100 °C for ≥0.5 min for viscera. At treatments at 70 °C, the Td-values (3 log reduction time) were significantly lower (P abalone meat (6.07) and viscera (10.73). Td = 3 values were not significantly different (P > 0.05) between abalone meat (1.78) and abalone viscera (1.33) at treatments at 85 °C. This study suggests that 100 °C for ≥0.5 min could potentially be used to inactivate NoV in molluscan shellfishes, including viscera.

  20. Extending The Shelf Life Of Blood Platelets

    Science.gov (United States)

    Surgenor, Douglas M.

    1988-01-01

    New method of storing human blood platelets extends vitality for transfusions. Packaged as suspension in sterile liquid in plastic blood bags. Each bag placed between pair of plastic grids, and rubberbands placed around sandwich thus formed to hold together. Stored upright in open air or in container through which air pumped at rate of at least 45 L/min. Ensures that platelets receive ample oxygen and expiratory carbon dioxide form platelets removed before pH drops to harmful levels.

  1. Platelet Glycoprotein lb-1X and Malignancy

    Science.gov (United States)

    2011-09-01

    initiate coagulation, resulting in the formation of a fibrin - rich tumor cell- platelet emboli (Figure 1). Many of these coagulation factor-tumor cell...the tumor cell in a fibrin - rich web. (13;23;24) During this process, the platelet integrin receptor, aIIb 3, serves as a receptor linking fibrin ... platelets , and tumor cells into a fibrin rich clot normally associated with a thrombus. (25;25) Indeed, it can be speculated the fibrin - rich clot

  2. The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

    Directory of Open Access Journals (Sweden)

    Marlien Pieters

    Full Text Available Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g, platelet-containing (352 g and platelet-rich plasma (200 g were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation. Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly

  3. Platelet aggregation and quality control of platelet concentrates produced in the Amazon Blood Bank

    Directory of Open Access Journals (Sweden)

    Maria José Dantas Coêlho

    2011-01-01

    Full Text Available BACKGROUND: The study of platelet aggregation is essential to assess in vitro platelet function by different platelet activation pathways. OBJECTIVE: To assess aggregation and biochemical parameters of random platelet concentrates produced at the Fundação HEMOAM using the quality control tests defined by law. METHODS: Whole blood samples from 80 donors and the respective platelet concentrate units were tested. Platelet concentrates were tested (platelet count, aggregation and pH on days 1, 3 and 5 of storage. Additionally a leukocyte count was done only on day 1 and microbiological tests on day 5 of storage. Collagen and adenosine diphosphate were used as inducing agonists for platelet aggregation testing. RESULTS: Donor whole blood had normal aggregation (aggregation with adenosine diphosphate = 67% and with collagen = 78%. The median aggregation in platelet concentrates with adenosine diphosphate was low throughout storage (18% on day 1, 7% on day 3 and 6% on day 5 and the median aggregation with collagen was normal only on day 1 and low thereafter (54.4% on day 1, 20.5% on day 3 and 9% on day 5. CONCLUSION: Although the results were within the norms required by law, platelet concentrates had low aggregation rates. We suggest the inclusion of a functional assessment test for the quality control of platelet concentrates for a more effective response to platelet replacement therapy.

  4. Reduced platelet-mediated and enhanced leukocyte-mediated fibrinolysis in experimentally induced diabetes in rats

    International Nuclear Information System (INIS)

    Winocour, P.D.; Colwell, J.A.

    1985-01-01

    Studies of fibrinolytic activity in diabetes mellitus have produced conflicting results. This may be a result of methodologic insensitivity or of variable contributions of the different blood components to whole blood fibrinolysis. To explore these two possibilities, the authors used a sensitive solid-phase radiometric assay to examine the fibrinolytic activity of whole blood, platelet-rich plasma, leukocytes, and platelet- and leukocyte-poor plasma prepared from control rats and rats with streptozocin-induced diabetes at various times after induction of diabetes. Fibrinolytic activity of whole blood from diabetic rats after 7 days was significantly reduced, and remained reduced after longer durations of diabetes up to 28 days. Platelet-rich plasma from diabetic rats had decreased fibrinolytic activity, which followed the same time course of changes as in whole blood. The platelet contribution to whole blood fibrinolysis was further reduced in vivo after 14 days of diabetes by a reduced whole blood platelet count. In contrast, fibrinolytic activity of leukocytes from diabetic rats became enhanced after 7 days of diabetes. After 49 days of diabetes, the whole blood leukocyte count was reduced, and in vivo would offset the enhanced activity. Plasma fibrinolytic activity was small compared with that of whole blood and was unaltered in diabetic rats. The authors conclude that altered platelet function contributes to decreased fibrinolytic activity of whole blood in diabetic rats, and that this may be partially offset by enhanced leukocyte-mediated fibrinolysis

  5. Description of a double centrifugation tube method for concentrating canine platelets.

    Science.gov (United States)

    Perazzi, Anna; Busetto, Roberto; Martinello, Tiziana; Drigo, Michele; Pasotto, Daniela; Cian, Francesco; Patruno, Marco; Iacopetti, Ilaria

    2013-07-22

    To evaluate the efficiency of platelet-rich plasma preparations by means of a double centrifugation tube method to obtain platelet-rich canine plasma at a concentration at least 4 times higher than the baseline value and a concentration of white blood cells not exceeding twice the reference range. A complete blood count was carried out for each sample and each concentrate. Whole blood samples were collected from 12 clinically healthy dogs (consenting blood donors). Blood was processed by a double centrifugation tube method to obtain platelet concentrates, which were then analyzed by a flow cytometry haematology system for haemogram. Platelet concentration and white blood cell count were determined in all samples. Platelet concentration at least 4 times higher than the baseline value and a white blood cell count not exceeding twice the reference range were obtained respectively in 10 cases out of 12 (83.3%) and 11 cases out of 12 (91.6%). This double centrifugation tube method is a relatively simple and inexpensive method for obtaining platelet-rich canine plasma, potentially available for therapeutic use to improve the healing process.

  6. BACTERIAL LYSATES FOR TOPICAL APPLICATION IN PREVENTION AND TREATMENT OF CHRONIC TONSILLITIS AMONG CHILDREN

    Directory of Open Access Journals (Sweden)

    T.I. Garashchenko

    2006-01-01

    Full Text Available The authors have carried on the research of the influence Imudon exerts (during topical application on the run of chronic tonsillitis among 48 children aged between 5 and 10 years old, being dispensary registrants. The sublingual application of a medicine 6 pills daily within 20 days demonstrated the frequency reduction of chronic tonsillitis acerbations by 2.9 times, as well as the reduction of total need in systemic antibacterial treatment by 10 times. Apart from that, the frequency of S. pyogenes group a exposure reduced by 3 times. The researchers noticed the tendency to normalization of pharynx biocenosis. Thus, Imudon may be recommended for the daily courses of treatment to the people, suffering from chronic tonsillitis, palatine tonsil auxesis and recurrent tonsillo-pharyngites.Key words: chronic tonsillitis, children, prevention, bacterial lysates.

  7. Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.

    Science.gov (United States)

    Metafora, S; Terada, M; Dow, L W; Marks, P A; Bank, A

    1972-05-01

    Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.

  8. Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

    Directory of Open Access Journals (Sweden)

    Andrew L Frelinger

    Full Text Available Activated autologous platelet-rich plasma (PRP used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma.To determine whether sub-microsecond duration, low electric field (SMLEF bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity.PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF, vascular endothelial growth factor (VEGF, endothelial growth factor (EGF and platelet factor 4 (PF4, and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin.PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet

  9. Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

    Science.gov (United States)

    Frelinger, Andrew L; Gerrits, Anja J; Garner, Allen L; Torres, Andrew S; Caiafa, Antonio; Morton, Christine A; Berny-Lang, Michelle A; Carmichael, Sabrina L; Neculaes, V Bogdan; Michelson, Alan D

    2016-01-01

    Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet

  10. Adjuvant treatment with the bacterial lysate (OM-85 improves management of atopic dermatitis: A randomized study.

    Directory of Open Access Journals (Sweden)

    Christine Bodemer

    Full Text Available Environmental factors play a major role on atopic dermatitis (AD which shows a constant rise in prevalence in western countries over the last decades. The Hygiene Hypothesis suggesting an inverse relationship between incidence of infections and the increase in atopic diseases in these countries, is one of the working hypothesis proposed to explain this trend.This study tested the efficacy and safety of oral administration of the bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®, in the treatment of established AD in children.Children aged 6 months to 7 years, with confirmed AD diagnosis, were randomized in a double-blind, placebo-controlled trial to receive, in addition to conventional treatment with emollients and topical corticosteroids, 3.5mg of the bacterial extract OM-85 or placebo daily for 9 months. The primary end-point was the difference between groups in the occurrence of new flares (NF during the study period, evaluated by Hazard Ratio (HR derived from conditional Cox proportional hazard regression models accounting for repeated events.Among the 179 randomized children, 170 were analysed, 88 in the OM-85 and 82 in the placebo group. As expected most children in both treatment groups experienced at least 1 NF during the study period (75 (85% patients in the OM-85 group and 72 (88% in the placebo group. Patients treated with OM-85 as adjuvant therapy had significantly fewer and delayed NFs (HR of repeated flares = 0.80; 95% confidence interval (CI: 0.67-0.96, also when potential confounding factors, as family history of atopy and corticosteroids use, were taken into account (HR = 0.82; 95% CI: 0.69-0.98. No major side effect was reported, with comparable and good tolerability for OM-85 and placebo.Results show an adjuvant therapeutic effect of a well standardized bacterial lysate OM-85 on established AD.

  11. Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor

    Czech Academy of Sciences Publication Activity Database

    Vaisocherová, Hana; Šípová, Hana; Víšová, Ivana; Bocková, Markéta; Špringer, Tomáš; Ermini, Maria Laura; Chadtová Song, Xue; Krejčík, Z.; Chrastinová, L.; Pastva, O.; Pimková, K.; Merkerová-Dostálová, M.; Dyr, J. E.; Homola, Jiří

    2015-01-01

    Roč. 70, August 05 (2015), s. 226-231 ISSN 0956-5663 R&D Projects: GA ČR(CZ) GBP205/12/G118 Institutional support: RVO:67985882 Keywords : Erythrocyte lysate * Polymer brushes * DNA array Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 7.476, year: 2015

  12. Activation of NO and cytokines by low-molecular weight fractions of lysates from G+ and G bacteria

    Czech Academy of Sciences Publication Activity Database

    Kmoníčková, Eva; Dusilová, Adéla; Kostecká, Petra; Kverka, Miloslav; Zídek, Zdeněk

    2013-01-01

    Roč. 157, Suppl.1 (2013), S51-S55 ISSN 1213-8118 R&D Projects: GA ČR GAP303/12/0535 Institutional support: RVO:68378041 ; RVO:61388971 Keywords : bacteria * lysate * immunomodulation Subject RIV: FR - Pharmacology ; Medidal Chemistry; EC - Immunology (MBU-M) Impact factor: 1.661, year: 2013

  13. Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

    Directory of Open Access Journals (Sweden)

    Yutaka Kitamura

    2018-02-01

    Full Text Available Platelet-rich fibrin (PRF clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

  14. Platelet function in the postprandial period

    Directory of Open Access Journals (Sweden)

    Sinzinger Helmut

    2012-09-01

    Full Text Available Abstract Background Postprandial hyperlipidemia and hyperglycemia have been related to cardiovascular events. Among different underlying mechanisms platelet activation seems to be responsible too. No comparable data between various tests in normo- vs. hyperlipidemics before and at different time intervals are available after a fat meal. We aimed to compare 9 of them within the same patients at several time points in postprandial hyperlipidemia. Results For some tests baseline values between the groups were significantly different (TXB2, platelet sensitivity, sedimentation and WU-test. However, hyperlipidemia revealed a variable influence on the tests examined. Some of the available tests apparently sensitive to show platelet activation reflect the increase in triglycerides (TG, such as the sedimentation index. ADP-induced platelet aggregatory activity in count adjusted washed isolated platelet samples during postprandial hyperlipidemia indicates mildly enhanced platelet activity, but does not seem to induce significant changes in aggregation. In patients with severe hypertriglyceridemia (> 400 mg/dl fasting changes in platelet function are more pronounced due to delayed decay and may last up to 16 hours paralleling TG reaching the prevalue. The overwhelming majority of platelet function tests do not significantly respond to postprandial hyperlipidemia. The correlation between the tests applied is poor. For standardization purpose, platelet aggregation tests, aimed to examine proaggregatory capacity in atherosclerosis, should only be performed at the same time of the day after a fasting period > 6 hours. The great variation in preanalytical work-up on comparison of various tests, large number of platelet tests available and their respective potential value are discussed. Conclusions At present, the suspicion that platelet function is significantly activated in the postprandial period cannot be supported by any of the tests used. The

  15. Reference intervals for platelet aggregation assessed by multiple electrode platelet aggregometry

    DEFF Research Database (Denmark)

    Rubak, Peter; Villadsen, Kirsten; Hvas, Anne-Mette

    2012-01-01

    Abstract Introduction Analyses of platelet aggregation in hirudin whole blood using Multiplate® was validated. Reference intervals for the most commonly used agonists were established, and the association between platelet aggregation, age, gender and haematological values was analysed. Material...... and methods We included 121 healthy individuals to establish reference intervals and six healthy individuals for evaluation of the day-to-day variation. Platelet aggregation was evaluated on hirudin whole blood employing Multiplate® induced by arachidonic acid, ADP, collagen and ristocetin (RISTOlow...... after adjusting for age and gender except for RISTOhigh. A positive significant association was found between platelet count and platelet aggregation (p

  16. Platelet storage in Fresenius/NPBI polyolefin and BTHC-PVC bags: a direct comparison.

    Science.gov (United States)

    Hornsey, V S; McColl, K; Drummond, O; Macgregor, I R; Prowse, C V

    2008-08-01

    New platelet storage systems, such as changes in the plastic of the storage bags, require validation. In this study, pooled buffy coat platelets stored in Fresenius/NPBI polyolefin bags were compared with those stored in Fresenius/NPBI butyryl-trihexyl citrate (BTHC) plasticized polyvinyl chloride (PVC). The CompoSelect thrombocyte polishing filter system (1000 mL polyolefin bag) and the CompoStop F730 system (1300 mL BTHC-PVC bag) were used to prepare paired, plasma-suspended, buffy coat platelet concentrates. Samples were taken up to day 7 for in vitro analysis. In a separate experiment, 12 units were prepared using the CompoStop F730 system and samples taken after leucofiltration for FXIIa assay. By day 7, platelet concentrates stored in BTHC-PVC demonstrated significantly higher pH levels (7.32 +/- 0.05 vs. 7.26 +/- 0.05) and a greater degree of cell lysis as shown by increased lactate dehydrogenase levels (497 +/- 107 vs. 392 +/- 81 U L(-1)). The supernatants contained higher concentrations of soluble P-selectin and the chemokine 'regulated on activation, normal T-cell expressed and presumably secreted', which are released from the alpha-granules during activation. The ATP concentrations were significantly lower in BTHC-PVC. Platelet counts, mean platelet volume and hypotonic shock response were similar for both bags. FXIIa antigen concentrations were 0.6 +/- 0.2 ng mL(-1) indicating that activation of the contact factor pathway had not occurred. Although the CompoStop F730 leucoreduction filter did not activate the contact system, platelets stored in 100% plasma in BTHC-PVC bags demonstrated different in vitro characteristics from those stored in polyolefin. Further work is required to demonstrate whether these differences will affect in vivo recovery and survival.

  17. Platelet-rich plasma for bone healing and regeneration.

    Science.gov (United States)

    Oryan, Ahmad; Alidadi, Soodeh; Moshiri, Ali

    2016-01-01

    Successful healing of large bone defects (LBDs) is a complicated phenomenon because the body's natural ability often fails to effectively repair the LBDs. New modalities should be utilized to increase the quality and accelerate bone healing. Platelet concentrates in different forms can be considered an attractive option for such purpose. Platelets as a natural source of growth factors, cytokines, and other micro and macromolecules are hypothesized to improve bone healing. This review has covered important concepts regarding platelet-rich plasma (PRP) including mechanisms of action, preparation protocols and their differences, and factors affecting the PRP efficacy during bone healing. In addition, the most recent studies in different levels which evaluated the role of PRP on bone repair has been reviewed and discussed to clarify the controversies and conflicts, and to illustrate a future prospective and directions for orthopedic surgeons to overcome current limitations and difficulties. As the efficacy of PRP is dependent on various factors, the outcome of PRP therapy is variable and unpredictable in orthopedic patients. Therefore, it is still too soon to suggest PRP as the first line treatment option in complicated bone injuries such as LBDs and nonunions. However, combination of PRP with natural and synthetic biomaterials can enhance the effectiveness of PRP.

  18. Automated processing of leucocyte-poor platelet concentrates.

    Science.gov (United States)

    Tandy, N P; Seghatchian, M J; Bessos, H

    1992-10-01

    In view of transfusion reactions and alloimmunization associated with leucocyte contamination of platelet concentrates (PC), there is a general move towards the production of leuco-poor PC. This goal is currently pursued by the production of various PC using buffy coat and apheresis techniques. Although there is no overall consensus on the meaning of 'leuco-poor', by assuming that this refers to a level of 5-50 x 10(7) leucocytes per PC, we were able to make comparisons with available systems used in Europe. In addition to platelet and white cell counts, other markers of PC quality were assessed in some cases. These included traditional markers (such as hypotonic stress response, pH, and lactate and beta-thromboglobulin levels) and newer markers (such as glycocalicin and plasma von Willebrand factor levels). Our preliminary results showed appreciable differences in platelet and white cell content of PC prepared by various types of apheresis equipment. Appreciable differences in the quality of stored PC were also observed between routine PC (non-leuco-poor and buffy coat and apheresed PC (leuco-poor).

  19. Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

    OpenAIRE

    Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-hara, Tomoko; Fujita, Naoya

    2014-01-01

    The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the pro...

  20. Platelet-rich plasma: why intra-articular? A systematic review of preclinical studies and clinical evidence on PRP for joint degeneration.

    Science.gov (United States)

    Filardo, G; Kon, E; Roffi, A; Di Matteo, B; Merli, M L; Marcacci, M

    2015-09-01

    The aim of this review was to analyze the available evidence on the clinical application of this biological approach for the injective treatment of cartilage lesions and joint degeneration, together with preclinical studies to support the rationale for the use of platelet concentrates, to shed some light and give indications on what to treat and what to expect from intra-articular injections of platelet-rich plasma (PRP). All in vitro, in vivo preclinical and clinical studies on PRP injective treatment in the English language concerning the effect of PRP on cartilage, synovial tissue, menisci, and mesenchymal stem cells were considered. A systematic review on the PubMed database was performed using the following words: (platelet-rich plasma or PRP or platelet concentrate or platelet lysate or platelet supernatant) and (cartilage or chondrocytes or synoviocytes or menisci or mesenchymal stem cells). Fifty-nine articles met the inclusion criteria: 26 were in vitro, 9 were in vivo, 2 were both in vivo and in vitro, and 22 were clinical studies. The analysis showed an increasing number of published studies over time. Preclinical evidence supports the use of PRP injections that might promote a favourable environment for joint tissues healing. Only a few high-quality clinical trials have been published, which showed a clinical improvement limited over time and mainly documented in younger patients not affected by advanced knee degeneration. Besides the limits and sometimes controversial findings, the preclinical literature shows an overall support toward this PRP application. An intra-articular injection does not just target cartilage; instead, PRP might influence the entire joint environment, leading to a short-term clinical improvement. Many biological variables might influence the clinical outcome and have to be studied to optimize PRP injective treatment of cartilage degeneration and osteoarthritis.

  1. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF).

    Science.gov (United States)

    Masuki, Hideo; Okudera, Toshimitsu; Watanebe, Taisuke; Suzuki, Masashi; Nishiyama, Kazuhiko; Okudera, Hajime; Nakata, Koh; Uematsu, Kohya; Su, Chen-Yao; Kawase, Tomoyuki

    2016-12-01

    The development of platelet-rich fibrin (PRF) drastically simplified the preparation procedure of platelet-concentrated biomaterials, such as platelet-rich plasma (PRP), and facilitated their clinical application. PRF's clinical effectiveness has often been demonstrated in pre-clinical and clinical studies; however, it is still controversial whether growth factors are significantly concentrated in PRF preparations to facilitate wound healing and tissue regeneration. To address this matter, we performed a comparative study of growth factor contents in PRP and its derivatives, such as advanced PRF (A-PRF) and concentrated growth factors (CGF). PRP and its derivatives were prepared from the same peripheral blood samples collected from healthy donors. A-PRF and CGF preparations were homogenized and centrifuged to produce extracts. Platelet and white blood cell counts in A-PRF and CGF preparations were determined by subtracting those counts in red blood cell fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts of whole blood samples. Concentrations of growth factors (TGF-β1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1β, IL-6) were determined using ELISA kits. Compared to PRP preparations, both A-PRF and CGF extracts contained compatible or higher levels of platelets and platelet-derived growth factors. In a cell proliferation assay, both A-PRF and CGF extracts significantly stimulated the proliferation of human periosteal cells without significant reduction at higher doses. These data clearly demonstrate that both A-PRF and CGF preparations contain significant amounts of growth factors capable of stimulating periosteal cell proliferation, suggesting that A-PRF and CGF preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application.

  2. Evolution, current status and advances in application of platelet concentrate in periodontics and implantology.

    Science.gov (United States)

    Agrawal, Amit Arvind

    2017-05-16

    Platelet concentrates (PC) [platelet-rich plasma (PRP) and platelet-rich fibrin (PRF)] are frequently used for surgical procedures in medical and dental fields, particularly in oral and maxillofacial surgery, plastic surgery and sports medicine. The objective of all these technologies is to extract all the elements from a blood sample that could be used to improve healing and promote tissue regeneration. Although leukocyte rich and leukocyte poor PRP's have their own place in literature, the importance of non-platelet components in a platelet concentrate remains a mystery. PC have come a long way since its first appearance in 1954 to the T-PRF, A-PRF and i-PRF introduced recently. These PC find varied applications successfully in periodontics and implant dentistry as well. However, the technique of preparation, standing time, transfer process, temperature of centrifuge, vibration, etc ., are the various factors for the mixed results reported in the literature. Until the introduction of a proper classification of terminologies, the PC were known by different names in different countries and by different commercial companies which also created a lot of confusion. This review intends to clarify all these confusion by briefing the exact evolution of PC, their preparation techniques, recent advances and their various clinical and technical aspects and applications.

  3. Intranasal Coadministration of the Cry1Ac Protoxin with Amoebal Lysates Increases Protection against Naegleria fowleri Meningoencephalitis

    Science.gov (United States)

    Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A.; López-Revilla, Rubén; Reséndiz-Albor, Aldo A.; Moreno-Fierros, Leticia

    2004-01-01

    Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 × 104 live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines. PMID:15271892

  4. Superior integrin activating capacity and higher adhesion to fibrinogen matrix in buffy coat-derived platelet concentrates (PCs) compared to PRP-PCs.

    Science.gov (United States)

    Beshkar, Pezhman; Hosseini, Ehteramolsadat; Ghasemzadeh, Mehran

    2018-02-01

    Regardless of different sources, methods or devices which are applied for preparation of therapeutic platelets, these products are generally isolated from whole blood by the sedimentation techniques which are based on PRP or buffy coat (BC) separation. As a general fact, platelet preparation and storage are also associated with some deleterious changes that known as platelet storage lesion (PSL). Although these alternations in platelet functional activity are aggravated during storage, whether technical issues within preparation can affect integrin activation and platelet adhesion to fibrinogen were investigated in this study. PRP- and BC-platelet concentrates (PCs) were subjected to flowcytometry analysis to examine the expression of platelet activation marker, P-selectin as well as active confirmation of the GPIIb/IIIa (α IIb β 3 ) on day 0, 1, 3 and 5 post-storage. Platelet adhesion to fibrinogen matrix was evaluated by fluorescence microscopy. Glucose concentration and LDH activity were also measured by colorimetric methods. The increasing P-selectin expression during storage was in a reverse correlation with PAC-1 binding (r = -0.67; p = .001). PRP-PCs showed the higher level of P-selectin expression than BC-PCs, whereas the levels of PAC-1 binding and platelet adhesion to fibrinogen matrix were significantly lower in PRP-PCs. Higher levels of active confirmation of the GPIIb/IIIa in BC-PCs were also associated with greater concentration of glucose in these products. We demonstrated the superior capacities of integrin activation and adhesion to fibrinogen for BC-PCs compared to those of PRP-PCs. These findings may provide more advantages for BC method of platelet preparation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Encapsulation of Gibbsite platelets with free radical and controlled radical emulsion polymerization approaches, a small review

    NARCIS (Netherlands)

    Loiko, O.P.; Spoelstra, A.B.; van Herk, A.M.; Meuldijk, J.; Heuts, J.P.A.

    2016-01-01

    Water-borne anisotropic polymer-Gibbsite latex particles were prepared by a conventional and an atom transfer radical polymerisation (ATRP) based starved-feed emulsion polymerisation without any chemical modification of the platelet surface. Anionic co-oligomers, synthesised via ATRP, were used in

  6. The biology of platelet-rich plasma and its application in oral surgery: literature review.

    NARCIS (Netherlands)

    Nikolidakis, D.; Jansen, J.A.

    2008-01-01

    Platelet-rich plasma (PRP) is a new approach in tissue regeneration and a developing area for clinicians and researchers. It is used in various surgical fields, including oral and maxillofacial surgery. PRP is prepared from the patient's own blood and contains growth factors that influence wound

  7. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

    NARCIS (Netherlands)

    Naaijkens, B.A.; Niessen, H.W.M.; Prins, H.J.; Krijnen, P.A.J.; Kokhuis, T.J.A.; de Jong, N.; van Hinsbergh, V.W.M.; Kamp, O.; Helder, M.N.; Musters, R.J.P.; van Dijk, A.; Juffermans, L.J.M.

    2012-01-01

    Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

  8. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

    NARCIS (Netherlands)

    B. Naaijkens (Benno); H.W.M. Niessen (Hans ); H.-J. Prins (H.); P.A.J. Krijnen (Paul); T.J.A. Kokhuis (Tom); N. de Jong (Nico); V.W.M. van Hinsbergh (Victor); O. Kamp (Otto); K. Helder MScN (Onno); R.J.P. Musters (René); A. van Dijk (Annemieke); L.J.M. Juffermans (Lynda)

    2012-01-01

    textabstractAdipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

  9. Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue–Derived Human Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Galeano-Garces, Catalina; Camilleri, Emily T.; Riester, Scott M.; Dudakovic, Amel; Larson, Dirk R.; Qu, Wenchun; Smith, Jay; Dietz, Allan B.; Im, Hee-Jeong; Krych, Aaron J.; Larson, A. Noelle; Karperien, Marcel; van Wijnen, Andre J.

    2017-01-01

    Objective: To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue–derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Design: Both

  10. The effect of storage on platelet morphology

    NARCIS (Netherlands)

    Sturk, A.; Burt, L. M.; Hakvoort, T.; ten Cate, J. W.; Crawford, N.

    1982-01-01

    Platelet concentrates were stored for one, two or three days at 4 degrees C (unagitated) or at room temperature (unagitated and linearly agitated). After washing the concentrates twice at room temperature and then incubating them for 60 minutes at 37 degrees C, the platelet morphology was

  11. Novel agents for anti-platelet therapy

    Directory of Open Access Journals (Sweden)

    Ji Xuebin

    2011-11-01

    Full Text Available Abstract Anti-platelet therapy plays an important role in the treatment of patients with thrombotic diseases. The most commonly used anti-platelet drugs, namely, aspirin, ticlopidine, and clopidogrel, are effective in the prevention and treatment of cardio-cerebrovascular diseases. Glycoprotein IIb/IIIa antagonists (e.g., abciximab, eptifibatide and tirofiban have demonstrated good clinical benefits and safety profiles in decreasing ischemic events in acute coronary syndrome. However, adverse events related to thrombosis or bleeding have been reported in cases of therapy with glycoprotein IIb/IIIa antagonists. Cilostazol is an anti-platelet agent used in the treatment of patients with peripheral ischemia, such as intermittent claudication. Presently, platelet adenosine diphosphate P2Y(12 receptor antagonists (e.g., clopidogrel, prasugrel, cangrelor, and ticagrelor are being used in clinical settings for their pronounced protective effects. The new protease-activated receptor antagonists, vorapaxar and atopaxar, potentially decrease the risk of ischemic events without significantly increasing the rate of bleeding. Some other new anti-platelet drugs undergoing clinical trials have also been introduced. Indeed, the number of new anti-platelet drugs is increasing. Consequently, the efficacy of these anti-platelet agents in actual patients warrants scrutiny, especially in terms of the hemorrhagic risks. Hopefully, new selective platelet inhibitors with high anti-thrombotic efficiencies and low hemorrhagic side effects can be developed.

  12. The life cycle of platelet granules.

    Science.gov (United States)

    Sharda, Anish; Flaumenhaft, Robert

    2018-01-01

    Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans -Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

  13. The origin and function of platelet glycosyltransferases

    DEFF Research Database (Denmark)

    Wandall, Hans H; Rumjantseva, Viktoria; Sørensen, Anne Louise Tølbøll

    2012-01-01

    Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and anti-angiogenic factors throughout the vascular system. Platelets are anucleated cells and lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartme...

  14. The interplay between platelets and coagulation

    NARCIS (Netherlands)

    Weeterings, C.

    2009-01-01

    Platelet activation and blood coagulation are two processes often studied separately, but which cannot be seen independently from each other. Platelets play a pivotal role in coagulation, not only by providing negatively charged phospholipids, but also in localizing the coagulation process from a

  15. 21 CFR 864.6675 - Platelet aggregometer.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Platelet aggregometer. 864.6675 Section 864.6675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675 Platelet...

  16. Platelet transfusion therapy: from 1973 to 2005.

    NARCIS (Netherlands)

    Brand, A.; Novotny, V.M.J.; Tomson, B.

    2006-01-01

    Platelet transfusions are indispensable for supportive care of patients with hematological diseases. We describe the developments in platelet products for transfusion since the 1970s, when, in particular, support for patients with allo-antibodies against human leukocyte antigens was a laborious

  17. Application of platelet-rich plasma and platelet-rich fibrin in fat grafting: basic science and literature review.

    Science.gov (United States)

    Liao, Han-Tsung; Marra, Kacey G; Rubin, J Peter

    2014-08-01

    Due to the natural properties of fat, fat grafting remains a popular procedure for soft tissue volume augmentation and reconstruction. However, clinical outcome varies and is technique dependent. <