Publications by authors named "Asmita Banerjee"

21 Publications

  • Page 1 of 1

Sub-Regional Differences of the Human Amniotic Membrane and Their Potential Impact on Tissue Regeneration Application.

Front Bioeng Biotechnol 2020 13;8:613804. Epub 2021 Jan 13.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria.

For more than 100 years, the human amniotic membrane (hAM) has been used in multiple tissue regeneration applications. The hAM consists of cells with stem cell characteristics and a rich layer of extracellular matrix. Undoubtedly, the hAM with viable cells has remarkable properties such as the differentiation potential into all three germ layers, immuno-modulatory, and anti-fibrotic properties. At first sight, the hAM seems to be one structural entity. However, by integrating its anatomical location, the hAM can be divided into placental, reflected, and umbilical amniotic membrane. Recent studies show that cells of these amniotic sub-regions differ considerably in their properties such as morphology, structure, and content/release of certain bioactive factors. The aim of this review is to summarize these findings and discuss the relevance of these different properties for tissue regeneration. In summary, reflected amnion seems to be more immuno-modulatory and could have a higher reprogramming efficiency, whereas placental amnion seems to be pro-inflammatory, pro-angiogenic, with higher proliferation and differentiation capacity (e.g., chondrogenic and osteogenic), and could be more suitable for certain graft constructions. Therefore, we suggest that the respective hAM sub-region should be selected in consideration of its desired outcome. This will help to optimize and fine-tune the clinical application of the hAM.
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http://dx.doi.org/10.3389/fbioe.2020.613804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839410PMC
January 2021

Perinatal Derivatives: Where Do We Stand? A Roadmap of the Human Placenta and Consensus for Tissue and Cell Nomenclature.

Front Bioeng Biotechnol 2020 17;8:610544. Epub 2020 Dec 17.

Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy.

Progress in the understanding of the biology of perinatal tissues has contributed to the breakthrough revelation of the therapeutic effects of perinatal derivatives (PnD), namely birth-associated tissues, cells, and secreted factors. The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and characterization and clinical application of PnD.
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http://dx.doi.org/10.3389/fbioe.2020.610544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773933PMC
December 2020

Effect of Diphenyleneiodonium Chloride on Intracellular Reactive Oxygen Species Metabolism with Emphasis on NADPH Oxidase and Mitochondria in Two Therapeutically Relevant Human Cell Types.

Pharmaceutics 2020 Dec 23;13(1). Epub 2020 Dec 23.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in the AUVA Trauma Research Center, Austrian Cluster for Tissue Regeneration, A-1200 Vienna, Austria.

Reactive oxygen species (ROS) have recently been recognized as important signal transducers, particularly regulating proliferation and differentiation of cells. Diphenyleneiodonium (DPI) is known as an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) and is also affecting mitochondrial function. The aim of this study was to investigate the effect of DPI on ROS metabolism and mitochondrial function in human amniotic membrane mesenchymal stromal cells (hAMSCs), human bone marrow mesenchymal stromal cells (hBMSCs), hBMSCs induced into osteoblast-like cells, and osteosarcoma cell line MG-63. Our data suggested a combination of a membrane potential sensitive fluorescent dye, tetramethylrhodamine methyl ester (TMRM), and a ROS-sensitive dye, CM-H2DCFDA, combined with a pretreatment with mitochondria-targeted ROS scavenger MitoTEMPO as a good tool to examine effects of DPI. We observed critical differences in ROS metabolism between hAMSCs, hBMSCs, osteoblast-like cells, and MG-63 cells, which were linked to energy metabolism. In cell types using predominantly glycolysis as the energy source, such as hAMSCs, DPI predominantly interacted with NOX, and it was not toxic for the cells. In hBMSCs, the ROS turnover was influenced by NOX activity rather than by the mitochondria. In cells with aerobic metabolism, such as MG 63, the mitochondria became an additional target for DPI, and these cells were prone to the toxic effects of DPI. In summary, our data suggest that undifferentiated cells rather than differentiated parenchymal cells should be considered as potential targets for DPI.
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http://dx.doi.org/10.3390/pharmaceutics13010010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823933PMC
December 2020

Critical Impact of Human Amniotic Membrane Tension on Mitochondrial Function and Cell Viability In Vitro.

Cells 2019 12 15;8(12). Epub 2019 Dec 15.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Donaueschingenstraße 13, 1200 Vienna, Austria.

Amniotic cells show exciting stem cell features, which has led to the idea of using living cells of human amniotic membranes (hAMs) in toto for clinical applications. However, under common cell culture conditions, viability of amniotic cells decreases rapidly, whereby reasons for this decrease are unknown so far. Recently, it has been suggested that loss of tissue tension in vivo leads to apoptosis. Therefore, the aim of this study was to investigate the effect of tissue distention on the viability of amniotic cells in vitro. Thereby, particular focus was put on vital mitochondria-linked parameters, such as respiration and ATP synthesis. Biopsies of hAMs were incubated for 7-21 days either non-distended or distended. We observed increased B-cell lymphoma 2-associated X protein (BAX)/B-cell lymphoma (BCL)-2 ratios in non-distended hAMs at day seven, followed by increased caspase 3 expression at day 14, and, consequently, loss of viability at day 21. In contrast, under distention, caspase 3 expression increased only slightly, and mitochondrial function and cellular viability were largely maintained. Our data suggest that a mechano-sensing pathway may control viability of hAM cells by triggering mitochondria-mediated apoptosis upon loss of tension in vitro. Further studies are required to elucidate the underlying molecular mechanisms between tissue distention and viability of hAM cells.
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http://dx.doi.org/10.3390/cells8121641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953074PMC
December 2019

Isotopic constraint on the twentieth-century increase in tropospheric ozone.

Nature 2019 06 12;570(7760):224-227. Epub 2019 Jun 12.

Université Grenoble Alpes, CNRS, Institut des Géosciences de l'Environnement, Grenoble, France.

Tropospheric ozone (O) is a key component of air pollution and an important anthropogenic greenhouse gas. During the twentieth century, the proliferation of the internal combustion engine, rapid industrialization and land-use change led to a global-scale increase in O concentrations; however, the magnitude of this increase is uncertain. Atmospheric chemistry models typically predict an increase in the tropospheric O burden of between 25 and 50 per cent since 1900, whereas direct measurements made in the late nineteenth century indicate that surface O mixing ratios increased by up to 300 per cent over that time period. However, the accuracy and diagnostic power of these measurements remains controversial. Here we use a record of the clumped-isotope composition of molecular oxygen (OO in O) trapped in polar firn and ice from 1590 to 2016 AD, as well as atmospheric chemistry model simulations, to constrain changes in tropospheric O concentrations. We find that during the second half of the twentieth century, the proportion of OO in O decreased by 0.03 ± 0.02 parts per thousand (95 per cent confidence interval) below its 1590-1958 AD mean, which implies that tropospheric O increased by less than 40 per cent during that time. These results corroborate model predictions of global-scale increases in surface pollution and vegetative stress caused by increasing anthropogenic emissions of O precursors. We also estimate that the radiative forcing of tropospheric O since 1850 AD is probably less than +0.4 watts per square metre, consistent with results from recent climate modelling studies.
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http://dx.doi.org/10.1038/s41586-019-1277-1DOI Listing
June 2019

Oxygen Tension Strongly Influences Metabolic Parameters and the Release of Interleukin-6 of Human Amniotic Mesenchymal Stromal Cells .

Stem Cells Int 2018 28;2018:9502451. Epub 2018 Oct 28.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Donaueschingenstraße 13, 1200 Vienna, Austria.

The human amniotic membrane (hAM) has been used for tissue regeneration for over a century. (), cells of the hAM are exposed to low oxygen tension (1-4% oxygen), while the hAM is usually cultured in atmospheric, meaning high, oxygen tension (20% oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5% and 20% oxygen. We found 20% oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS); however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20% oxygen. To conclude, short-time cultivation at 20% oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy.
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http://dx.doi.org/10.1155/2018/9502451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230389PMC
October 2018

Cardiac Restoration Stemming From the Placenta Tree: Insights From Fetal and Perinatal Cell Biology.

Front Physiol 2018 11;9:385. Epub 2018 Apr 11.

Centro di Ricerca E. Menni, Fondazione Poliambulanza - Istituto Ospedaliero, Brescia, Italy.

Efficient cardiac repair and ultimate regeneration still represents one of the main challenges of modern medicine. Indeed, cardiovascular disease can derive from independent conditions upsetting heart structure and performance: myocardial ischemia and infarction (MI), pharmacological cardiotoxicity, and congenital heart defects, just to name a few. All these disorders have profound consequences on cardiac tissue, inducing the onset of heart failure over time. Since the cure is currently represented by heart transplantation, which is extremely difficult due to the shortage of donors, much effort is being dedicated to developing innovative therapeutic strategies based on stem cell exploitation. Among the broad scenario of stem/progenitor cell subpopulations, fetal and perinatal sources, namely amniotic fluid and term placenta, have gained interest due to their peculiar regenerative capacity, high self-renewal capability, and ease of collection from clinical waste material. In this review, we will provide the state-of-the-art on fetal perinatal stem cells for cardiac repair and regeneration. We will discuss different pathological conditions and the main therapeutic strategies proposed, including cell transplantation, putative paracrine therapy, reprogramming, and tissue engineering approaches.
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http://dx.doi.org/10.3389/fphys.2018.00385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904405PMC
April 2018

Cellular and Site-Specific Mitochondrial Characterization of Vital Human Amniotic Membrane.

Cell Transplant 2018 01;27(1):3-11

1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria.

Over a century ago, clinicians started to use the human amniotic membrane for coverage of wounds and burn injuries. To date, literally thousands of different clinical applications exist for this biomaterial almost exclusively in a decellularized or denuded form. Recent reconsiderations for the use of vital human amniotic membrane for clinical applications would take advantage of the versatile cells of embryonic origin including the entirety of their cell organelles. Recently, more and more evidence was found, showing mitochondria to be involved in most fundamental cellular processes, such as differentiation and cell death. In this study, we focused on specific properties of mitochondria of vital human amniotic membrane and characterized bioenergetical parameters of 2 subregions of the human amniotic membrane, the placental and reflected amnion. We found significantly different levels of adenosine triphosphate (ATP) and extracellular reactive oxygen species, concentrations of succinate dehydrogenase, and lactate upon inhibition of ATP synthase in placental and reflected amnion. We also found significantly different rates of mitochondrial respiration in isolated human amniotic epithelial cells and human amniotic mesenchymal stromal cells, according to the subregions. Differences in metabolic activities were inversely related to mitochondrial DNA copy numbers in isolated cells of placental and reflected amnion. Based on significant differences of several key parameters of energy metabolism in 2 subregions of vital amnion, we propose that these metabolic differences of vital placental and reflected amnion could have critical impact on therapeutic applications. Inclusion of region-specific metabolic properties could optimize and fine-tune the clinical application of the human amniotic membrane and improve the outcome significantly.
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http://dx.doi.org/10.1177/0963689717735332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434485PMC
January 2018

Secretome Conveys the Protective Effects of ASCs: Therapeutic Potential Following Hemorrhagic Shock?

Shock 2018 10;50(4):442-448

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in the AUVA Research Center, Vienna, Austria.

Objectives: We tested whether resuscitation supplemented with rat adipose-derived stem cells (ASCs) or secretome (conditioned media) of ASCs can ameliorate inflammation, cell/organ injury, and/or improve outcome after hemorrhagic traumatic shock (HTS).

Interventions: Rats were subjected to HTS and a resuscitation protocol that mimics prehospital restrictive reperfusion followed by an adequate reperfusion phase. Twenty minutes into the restrictive reperfusion, animals received an intravenous bolus of 2 × 10 cells (ASC group) or the secretome produced by 2 × 10 ASCs/24 h (ASC-Secretome group). Controls received the vehicle (Vehicle group). All rats were observed for 28-day survival.

Measurements And Main Results: HTS-induced inflammation represented by IL-6 was inhibited in the ASC (80%, P < 0.001) and in ASC-Secretome (59%, P < 0.01) group at 48 h compared with Vehicle group. At 24 h, HTS-induced liver injury reflected in plasma alanine aminotransferase was ameliorated by 36% (P < 0.001) in both the ASC and ASC-Secretome groups when compared with the Vehicle. There was no effect on kidney function and/or general cell injury markers. HTS induced a moderate 28-day mortality (18%) that was prevented (P = 0.08) in the ASC but not in the ASC-Secretome group (12%).

Conclusions: Our data suggest that the ASC-secretome supplemented resuscitation following HTS, in the absence of the stem cells, exerts anti-inflammatory and liver protective effects. Given its ease of preparation, storage, availability, and application (in contrast to the stem cells) we believe that the cell-free secretome has a better therapeutic potential in the early phase of an acute hemorrhagic shock scenario.
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http://dx.doi.org/10.1097/SHK.0000000000001047DOI Listing
October 2018

Different metabolic activity in placental and reflected regions of the human amniotic membrane.

Placenta 2015 Nov 5;36(11):1329-32. Epub 2015 Sep 5.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Donaueschingenstraße 13, 1200 Vienna, Austria; Austrian Cluster for Tissue Regeneration, Austria.

Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications.
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http://dx.doi.org/10.1016/j.placenta.2015.08.015DOI Listing
November 2015

Activated Schwann Cell-Like Cells on Aligned Fibrin-Poly(Lactic-Co-Glycolic Acid) Structures: A Novel Construct for Application in Peripheral Nerve Regeneration.

Cells Tissues Organs 2015 16;200(5):287-99. Epub 2015 Sep 16.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria.

Tissue engineering approaches in nerve regeneration search for ways to support gold standard therapy (autologous nerve grafts) and to improve results by bridging nerve defects with different kinds of conduits. In this study, we describe electrospinning of aligned fibrin-poly(lactic-co-glycolic acid) (PLGA) fibers in an attempt to create a biomimicking tissue-like material seeded with Schwann cell-like cells (SCLs) in vitro for potential use as an in vivo scaffold. Rat adipose-derived stem cells (rASCs) were differentiated into SCLs and evaluated with flow cytometry concerning their differentiation and activation status [S100b, P75, myelin-associated glycoprotein (MAG), and protein 0 (P0)]. After receiving the proliferation stimulus forskolin, SCLs expressed S100b and P75; comparable to native, activated Schwann cells, while cultured without forskolin, cells switched to a promyelinating phenotype and expressed S100b, MAG, and P0. Human fibrinogen and thrombin, blended with PLGA, were electrospun and the alignment and homogeneity of the fibers were proven by scanning electron microscopy. Electrospun scaffolds were seeded with SCLs and the formation of Büngner-like structures in SCLs was evaluated with phalloidin/propidium iodide staining. Carrier fibrin gels containing rASCs acted as a self-shaping matrix to form a tubular structure. In this study, we could show that rASCs can be differentiated into activated, proliferating SCLs and that these cells react to minimal changes in stimulus, switching to a promyelinating phenotype. Aligned electrospun fibrin-PLGA fibers promoted the formation of Büngner-like structures in SCLs, which also rolled the fibrin-PLGA matrix into a tubular scaffold. These in vitro findings favor further in vivo testing.
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http://dx.doi.org/10.1159/000437091DOI Listing
September 2016

Vicious inducible nitric oxide synthase-mitochondrial reactive oxygen species cycle accelerates inflammatory response and causes liver injury in rats.

Antioxid Redox Signal 2015 Mar 22;22(7):572-86. Epub 2014 Dec 22.

1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .

Aims: Increasing evidences suggest that, apart from activation of guanylyl cyclase, intracellular nitric oxide (NO) signaling is associated with an interaction between NO and reactive oxygen species (ROS) to modulate physiological or pathophysiological processes. The aim of this study was to understand the contribution of mitochondrial ROS (mtROS) to NO-mediated signaling in hepatocytes on inflammation.

Results: In rats treated with lipopolysaccharide (LPS), mitochondria-targeted antioxidants (mtAOX) (mitoTEMPO and SkQ1) reduced inducible nitric oxide synthase (iNOS) gene expression in liver, NO levels in blood and plasma, and markers of organ damage (lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase). In cultured hepatocytes, treated with inflammatory mediators, generated ex vivo by incubation of white blood cells with LPS, we observed an increase in NO and mtROS levels. l-NG-monomethyl arginine citrate, a NOS inhibitor, decreased both NO and mtROS levels. mtAOX reduced mtROS, cytoplasmic ROS levels, and expression of iNOS and interleukin (IL)-6. These data suggest that NO, generated by iNOS, elevates mtROS, which, in turn, diffuse into the cytoplasm and upregulate iNOS and IL-6.

Innovation: Here, for the first time, we show that intracellular signaling pathways mediated by NO and ROS are linked to each other via mtROS and form an iNOS-mtROS feed-forward loop which aggravates liver failure on acute inflammation.

Conclusion: Our results provide a mechanistic explanation of how NO and mtROS cooperate to conduct inflammatory intracellular signals. We anticipate our results to be the missing mechanistic link between acute systemic inflammation and liver failure.
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http://dx.doi.org/10.1089/ars.2014.5996DOI Listing
March 2015

In vitro extracorporeal shock wave treatment enhances stemness and preserves multipotency of rat and human adipose-derived stem cells.

Cytotherapy 2014 Dec 28;16(12):1666-78. Epub 2014 Aug 28.

Austrian Cluster for Tissue Regeneration, Vienna, Austria; University of Applied Sciences Technikum Wien-Department of Biochemical Engineering, Vienna, Austria.

Background Aims: Adipose-derived progenitor/stem cells (ASCs) are discussed as a promising candidate for various tissue engineering approaches. However, its applicability for the clinic is still difficult due to intra- and inter-donor heterogeneity and limited life span in vitro, influencing differentiation capacity as a consequence to decreased multipotency.

Methods: Extracorporeal shock wave treatment has been proven to be a suitable clinical tool to improve regeneration of a variety of tissues for several decades, whereas the mechanisms underlying these beneficial effects remain widely unknown.

Results: In this study we show that human and rat adipose derived stem cells respond strongly to repetitive shock wave treatment in vitro, resulting not only in maintenance and significant elevation of mesenchymal markers (CD73, CD90, CD105), but also in significantly increased differentiation capacity towards the osteogenic and adipogenic lineage as well as toward Schwann-cell like cells even after extended time in vitro, preserving multipotency of ASCs.

Conclusions: ESWT might be a promising tool to improve ASC quality for cell therapy in various tissue engineering and regenerative medicine applications.
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http://dx.doi.org/10.1016/j.jcyt.2014.07.005DOI Listing
December 2014

In toto differentiation of human amniotic membrane towards the Schwann cell lineage.

Cell Tissue Bank 2014 Jun 29;15(2):227-39. Epub 2013 Oct 29.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Donaueschingenstraße 13, 1200, Vienna, Austria.

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.
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http://dx.doi.org/10.1007/s10561-013-9401-1DOI Listing
June 2014

Impact of mitochondria on nitrite metabolism in HL-1 cardiomyocytes.

Front Physiol 2013 20;4:101. Epub 2013 May 20.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in AUVA research centre Vienna, Austria.

Apart from ATP synthesis mitochondria have many other functions, one being nitrite reductase activity. Nitric oxide (NO) released from nitrite has been shown to protect the heart from ischemia/reperfusion (I/R) injury in a cGMP-dependent manner. However, the exact impact of mitochondria on the release of NO from nitrite in cardiomyocytes is not completely understood. Besides mitochondria, a number of non-mitochondrial metalloproteins have been suggested to facilitate this process. The aim of this study was to investigate the impact of mitochondria on the bioactivation of nitrite in HL-1 cardiomyocytes. The levels of nitrosyl complexes of hemoglobin (NO-Hb) and cGMP levels were measured by electron spin resonance spectroscopy and enzyme immunoassay. In addition the formation of free NO was determined by confocal microscopy as well as intracellular nitrite and S-nitrosothiols by chemoluminescence analysis. NO was released from nitrite in cell culture in an oxygen-dependent manner. Application of specific inhibitors of the respiratory chain, p450, NO synthases (NOS) and xanthine oxidoreductase (XOR) showed that all four enzymatic systems are involved in the release of NO, but more than 50% of NO is released via the mitochondrial pathway. Only NO released by mitochondria activated cGMP synthesis. Cardiomyocytes co-cultured with red blood cells (RBC) competed with RBC for nitrite, but free NO was detected only in HL-1 cells suggesting that RBC are not a source of NO in this model. Apart from activation of cGMP synthesis, NO formed in HL-1 cells diffused out of the cells and formed NO-Hb complexes. In addition nitrite was converted by HL-1 cells to S-nitrosyl complexes. In HL-1 cardiomyocytes, several enzymatic systems are involved in nitrite reduction to NO but only the mitochondrial pathway of NO release activates cGMP synthesis. Our data suggest that this pathway may be a key regulator of myocardial contractility especially under hypoxic conditions.
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http://dx.doi.org/10.3389/fphys.2013.00101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3657631PMC
June 2013

Enhanced reporter gene assay for the detection of osteogenic differentiation.

Tissue Eng Part C Methods 2011 Apr 14;17(4):401-10. Epub 2010 Dec 14.

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology/AUVA Research Center, The Austrian Cluster for Tissue Regeneration, European Institute of Excellence on Tissue Engineering and Regenerative Medicine Research (Expertissues EEIG), Vienna, Austria.

Detection of osteogenic differentiation is crucial for bone tissue engineering. Despite established standard end point assays, there is increasing demand for methods allowing noninvasive kinetic differentiation monitoring. Reporter gene assays employing tissue-specific promoters and suitable reporter genes fulfill these requirements. Many promoters, however, exhibit only weak cis-activating potential, thus limiting their application to generate sensitive reporter gene assays. Therefore, the aim of this study was to design a reporter gene assay employing elements of the murine osteocalcin promoter coupled to a viral enhancer for signal amplification. Additionally, the system's practicability was enhanced by introducing a secreted luciferase as a quantifiable reporter gene. The constructs were tested in C2C12 cells stimulated with recombinant human bone morphogenetic protein 2 for osteogenic differentiation in two-dimensional and three-dimensional culture. Osteogenic differentiation was confirmed by standard assays for osteogenesis. The reporter gene signal was detected through a secreted luciferase or fluorescence microscopy for enhanced yellow fluorescent protein. The constructs exhibited strong activation upon treatment with recombinant human bone morphogenetic protein 2. Weak background expression was observable in negative controls, attributed to the pan-active viral enhancer. In conclusion, a novel enhancer/tissue-specific promoter combination allows specific signal-amplified, kinetic monitoring of osteogenic differentiation in a nonsample-destructive manner.
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http://dx.doi.org/10.1089/ten.TEC.2010.0095DOI Listing
April 2011

Silk fibroin microparticles as carriers for delivery of human recombinant bone morphogenetic protein-2: in vitro and in vivo bioactivity.

Tissue Eng Part C Methods 2010 Oct;16(5):937-45

3B's Research Group-Biomaterials, Biodegradables, and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, Guimarães, Portugal.

The in vitro and in vivo efficiency of fibroin microparticles as a delivery carrier for bone morphogenetic protein-2 (BMP-2) was evaluated. BMP-2 was encapsulated in silk fibroin particles that were produced by a simple and very mild processing method. The dose-response of BMP-2-loaded fibroin particles was examined in C2C12 cells, after 5 days of culture. The BMP-2 retained most of its activity as observed by the increase in alkaline phosphatase activity, which was much higher when BMP-2 was encapsulated into the particles rather than just surface-adsorbed. After 2 weeks of culture, increased mineralization was observed with BMP-2-loaded particles in comparison to soluble added growth factor. No significant cytotoxicity was detected. When implanted in a rat ectopic model, bone formation was observed by in vivo micro-computed tomography after 2 and 4 weeks postimplantation, with particles loaded with 5 or 12.5 microg BMP-2. An increase in bone density was observed over time. Histology revealed further evidence of ectopic bone formation, observed by strong alizarin red staining and osteocalcin immunostaining. Our findings show that fibroin microparticles may present an interesting option for future clinical applications in the bone tissue engineering field, and therefore, further studies have been planned.
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http://dx.doi.org/10.1089/ten.TEC.2009.0486DOI Listing
October 2010

Thermoresponsive self-assembled elastin-based nanoparticles for delivery of BMPs.

J Control Release 2010 Mar 12;142(3):312-8. Epub 2009 Nov 12.

3B's Research Group, Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal.

Elastin-like polymers are a new type of protein-based polymers that display interesting properties in the biomaterial field. Bone morphogenetic proteins (BMPs) are cytokines with a strong ability to promote new bone formation. In this work, we explored the use of elastin-like nanoparticles (average size 237.5+/-3.0 nm), created by thermoresponsive self-assembly, for the combined release of bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-14 (BMP-14). These BMPs could be encapsulated at high efficiency into the elastin-like particles and delivered in a sustained way for 14 days. The activity of the growth factors was retained, as shown by the induction of ALP activity and osteogenic mineralization in C2C12 cells. Increased bioactivity was observed with a combined release of BMP-2 and BMP-14. This approach shows a significant potential for future tissue engineering applications in bone.
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http://dx.doi.org/10.1016/j.jconrel.2009.11.003DOI Listing
March 2010

Comparison of structure, strength and cytocompatibility of a fibrin matrix supplemented either with tranexamic acid or aprotinin.

J Biomed Mater Res B Appl Biomater 2007 Jul;82(1):109-14

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, The Research Center for Traumatology of the AUVA, Donaueschingenstrasse 13, A-1200 Vienna, Austria.

Fibrin sealants are used as hemostats, sealants, tissue adhesives, and as matrix for substances/cells in a number of surgical and tissue engineering procedures. Main characteristics of fibrin are high tensile strength, adhesive strength, biocompatibility, and resorption. A major adverse event would be premature fibrin lysis and recurrent bleeding. This must be prevented by fibrinolysis inhibitors. The most common fibrinolysis inhibitors used are aprotinin and tranexamic acid (t-AMCA). Comparison of commercially available fibrin sealants utilizing aprotinin or t-AMCA revealed a lower sealing efficacy in an in vivo lung resection model for a t-AMCA containing product. Therefore, we compared the influence of t-AMCA and aprotinin on structure, mechanical properties, and cytocompatibility of a fibrin matrix. In our experiments, we found that substitution of aprotinin with t-AMCA reduced the tensile strength and formation of fibrin fibers and affected viability of a fibroblast cell-line. In conclusion, t-AMCA negatively affects physical and biological properties of fibrin relevant for clinical application as well as tissue regeneration.
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http://dx.doi.org/10.1002/jbm.b.30711DOI Listing
July 2007

Expression and purification of biologically active rat bone morphogenetic protein-4 produced as inclusion bodies in recombinant Escherichia coli.

Biotechnol Lett 2005 Oct;27(20):1559-64

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, 1200, Vienna, Austria.

Rat bone morphogenetic protein-4 (rBMP-4) cDNA was cloned from rat osteoblasts by RT-PCR and expressed in E. coli. Monomeric, dimeric and polymeric forms of recombinant rat BMP-4 (rrBMP-4) were obtained from inclusion bodies after solubilization with urea. The dimer was separated from the remaining polymer and host cell contaminants using size exclusion chromatography. Furthermore, purified rrBMP-4 was stabilized at low urea concentration (40 mM) and at pH 8.5 through the addition of bovine serum albumin. Both, rrBMP-4 dimer and polymer were biologically active as tested by the induction of alkaline phosphatase activity in MC3T3-E1 cells.
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http://dx.doi.org/10.1007/s10529-005-1794-xDOI Listing
October 2005

Release of glutaraldehyde from an albumin-glutaraldehyde tissue adhesive causes significant in vitro and in vivo toxicity.

Ann Thorac Surg 2005 May;79(5):1522-8; discussion 1529

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Research Center for Traumatology AUVA, Vienna, Austria.

Background: A two-component sealant composed of bovine serum albumin and glutaraldehyde (BioGlue) is used to treat aortic dissections. Although glutaraldehyde guarantees strong adherence to tissues and synthetic materials, its toxic potential should be considered. The aim of this study was to determine the amount of glutaraldehyde released from BioGlue, its cytotoxic effects on cultured cells, and the local reaction of lung, liver, and aortic tissues to BioGlue.

Methods: BioGlue was prepared according to the product insert, allowed to polymerize, and then overlaid with saline solution. The supernatant was analyzed for its content of glutaraldehyde. The cytotoxic effect of BioGlue was evaluated by adding the supernatants to either cultured human embryo fibroblasts (MRC5) or mouse myoblasts (C2C12). In vivo toxicity was assessed on three different tissues by applying BioGlue onto a partial lung resection, a liver abrasion, or an intact abdominal aorta in rabbits. Tissue samples were histologically evaluated 2 and 7 days after application.

Results: Saline supernatants from polymerized BioGlue contained 100 to 200 mug/mL glutaraldehyde and were cytotoxic to both cell lines tested. Application of BioGlue to lung and liver tissue evoked serious adverse effects consisting of high-grade inflammation, edema, and toxic necrosis. Intact aortic tissue showed only low-grade or medium-grade inflammation.

Conclusions: Polymerized BioGlue releases amounts of glutaraldehyde that are capable of inducing cytotoxic effects both in vitro and in vivo. Use of BioGlue should be restricted to the aortic dissection procedure, as other tissues are sensitive to the amounts of glutaraldehyde released from the glue.
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http://dx.doi.org/10.1016/j.athoracsur.2004.11.054DOI Listing
May 2005
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