Comparison of the properties of mouse ES cells (mESCs), mouse epiblast stem cells (mEpiSCs), human ES cells (hESCs) and human iPS cells (hiPSCs).
\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
\n'}],latestNews:[{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"}]},book:{item:{type:"book",id:"1956",leadTitle:null,fullTitle:"Phytochemicals - Bioactivities and Impact on Health",title:"Phytochemicals",subtitle:"Bioactivities and Impact on Health",reviewType:"peer-reviewed",abstract:"Among the thousands of naturally occurring constituents so far identified in plants and exhibiting a long history of safe use, there are none that pose - or reasonably might be expected to pose - a significant risk to human health at current low levels of intake when used as flavoring substances. Due to their natural origin, environmental and genetic factors will influence the chemical composition of the plant essential oils. Factors such as species and subspecies, geographical location, harvest time, plant part used and method of isolation all affect chemical composition of the crude material separated from the plant. The screening of plant extracts and natural products for antioxidative and antimicrobial activity has revealed the potential of higher plants as a source of new agents, to serve the processing of natural products.",isbn:null,printIsbn:"978-953-307-424-5",pdfIsbn:"978-953-51-5194-4",doi:"10.5772/2373",price:139,priceEur:155,priceUsd:179,slug:"phytochemicals-bioactivities-and-impact-on-health",numberOfPages:402,isOpenForSubmission:!1,isInWos:1,hash:"bca0d717264e92e4863937bdcf16e06b",bookSignature:"Iraj Rasooli",publishedDate:"December 22nd 2011",coverURL:"https://cdn.intechopen.com/books/images_new/1956.jpg",numberOfDownloads:57743,numberOfWosCitations:70,numberOfCrossrefCitations:23,numberOfDimensionsCitations:96,hasAltmetrics:1,numberOfTotalCitations:189,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 3rd 2011",dateEndSecondStepPublish:"March 3rd 2011",dateEndThirdStepPublish:"July 8th 2011",dateEndFourthStepPublish:"August 7th 2011",dateEndFifthStepPublish:"December 5th 2011",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6",editedByType:"Edited by",kuFlag:!1,editors:[{id:"61446",title:"Prof.",name:"Iraj",middleName:null,surname:"Rasooli",slug:"iraj-rasooli",fullName:"Iraj Rasooli",profilePictureURL:"https://mts.intechopen.com/storage/users/61446/images/1890_n.jpg",biography:"Iraj Rasooli was born in 1960 in Ahar, Iran. He received his BSc in Microbiology from Shivaji University (India) before obtaining his MSc and PhD in 1992 and 1998 respectively in Microbiology from Bombay University (India). He joined Shahed University in 1993 as an assistant professor where his focus has been on research activities on biological properties of essential oils from medicinal plants and he has published a number of papers in a number of notable national and international journals. He was awarded Razi festival prize by the president of Islamic Republic of Iran in 2002. Professor Rasooli went to the University of Calgary (Canada) in 2002 for his sabbatical where he accelerated his level of knowledge of molecular biology at Prof. Anthony Schryver’s laboratory. To date he has published 86 full length papers on bioactivities of essential oils and molecular microbiology. 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Embryonic stem (ES) cells isolated from the inner cell mass (ICM) of blastocysts possess the defining pluroptency: unlimited self-renewal and giving rise to all cells of the organism[1, 2]. Thus, ES cells hold great promise for regenerative medicine to treat many diseases including heart failure, diabetes, Alzheimer’s and Parkinson’s disease by replacing the damaged cells with ES cell-derived healthy ones. The recent advent of induced pluripotent stem (iPS) cells reprogrammed from somatic cells has the potential to revolutionize the field of regenerative medicine since patient-derived iPS cells, in principle, circumvent the ethical problems and immune rejection associated with human ES cells[3]. Nevertheless, the future clinical translation of ES cells and iPS cells is facing numerous hurdles. Understanding the molecular mechanisms that impart ES cells with pluripotency may help address some of these challenges. The past few years have seen tremendous progress in understanding of mechanisms which govern ES cell pluripotency. In this chapter, we will review critical signaling and transcription factor networks that have been identified to maintain ES cell pluripotency.
ES cells require extrinsic growth factors to maintain their pluripotency in culture. These extrinsic growth factors act on different signaling pathways to regulate intrinsic transcription factor networks to sustain ES cells in the undifferentiated state. The signaling pathways required to support pluripotency in mouse ES cell are distinct from those in human ES cells (Figure 1).
Exogenous growth factors signal through distinct signaling pathways to regulate transcription factors for ES cell pluripotency.
Mouse ES cells were originally cultured on feeder layers derived from mouse embryonic fibroblasts (MEF). Later it was found that Leukaemia Inhibitory Factor (LIF), a member of the Interleukin-6 cytokines produced by MEFs, was the key factor to maintain pluripotency of mouse ES cells by inhibiting their differentiation[4]. Upon LIF binding, the LIF receptor recruits gp130 to form a heterodimer which subsequently activates Janus kinase (JAK) through transphosphorylation[5]. Activated JAK then phosphorylate gp130, creating a docking site to bind the SH2 domain of Signal Transducers and Activators of Transcription 3 (STAT3)[6-9]. Once STAT3 binds to the gp130 docking site, JAK then phosphorylates the recruited STAT3. Phosphorylated STAT3 forms a homodimer, which subsequently translocate into the nucleus, where it binds to gene enhancers to regulate target gene expression[10-12].
Although the LIF/JAK/STAT3 pathway has been well documented to maintain pluripotency of mouse ES cells in the presence of serum, the mechanisms by which activated STAT3 functions in this regard is poorly understood. Recently, studies in identification of STAT3 target genes have improved our understanding of activated STAT3 in maintaining pluripotency. Chen et al identified 718 STAT3-bound genomic sites that were co-occupied by pluripotency transcription markers (Oct4, Sox2 and Nanog) by using chromatin immunoprecipitation sequencing (ChIP-seq)[12]. In addition, Kidder and colleagues found that STAT3 target genes enriched in ES cells were downregulated in differentiated cells by mapping STAT3 binding targets in mouse ES cells and differentiated embryoid bodies (EBs)[13]. Along with these results, it has been demonstrated that knocking down STAT3-target genes induces activation of endodermal and mesodermal genes, supporting the conclusion that STAT3 prevents mESC differentiation by suppressing lineage-specific genes[14].
Interestingly, the LIF receptor and gp130 are also expressed in human ES cells and human LIF can induce STAT3 phosphorylation and nuclear translocation in human ES cells. However, human LIF is unable to maintain the pluripotent state of human ESs, suggesting that mouse and human ES cells require distinct signaling mechanisms to govern their pluripotency[15].
TGF-β superfamily consists of more than 40 members, including TGF-β, Activin, Nodal, and bone morphogenetic proteins (BMPs). The TGF-β members transduce signals by binding to heteromeric complexes of serine/threonine kinase receptors, type I and type II receptors, which subsequently activate intracellular Smad proteins. Smads 2 and 3 are specifically activated by activin, nodal and TGF-β ligands, whereas Smads 1, 5 and 8 are activated by BMP ligands[16, 17] (Figure 1). The TGF-β-related signaling pathways play complex roles in regulating the pluripotency and cell fate of ES cells.
Bone Morphogenetic Protein (BMP) is a subset of the TGF-β superfamily[18]. When BMP ligands bind to type II BMP receptors (BMPRII), BMPRII then recruits and phosphorylates type I BMP receptors (BMPRI). Activated type I receptors subsequently phosphorylate BMP-responsive SMAD1/5/8 which then forms a complex with SMAD4 and translocates into nucleus to regulate target gene expression (Figure 1). In mouse ES cells, LIF can substitute MEF feeder layers in maintaining pluripotency in the presence of animal serum by activating the transcription factor STAT3. However, in serum-free cultures, LIF is insufficient to block neural differentiation and maintain pluripotency. Recently, Ying et al reported that BMP was able to replace serum to maintain pluripotency of mouse ES cells in the presence of LIF. BMP has been shown to phosphorylate SMAD1/5 and activate inhibitors of differentiation (Id) genes, which block neural differentiation by antagonizing neurogenic transcription factors[19]. In the absence of MEF and serum, exogenous LIF, in combination with BMP4 proteins, can sufficiently maintain the pluripotency of mouse ES cells derived from “permissive” mouse strains.
In contrast to a maintenance role in mouse ES cell pluripotency, BMP has been shown to promote human ES cells differentiation to trophoblasts, and inhibiting BMP signaling with the BMP antagonist, Noggin, sustains the undifferentiated state of human ES cells[20, 21]. In consistence, dorsomorphin and DMH1, small molecule BMP inhibitors previously identified in our lab, were shown to promote long-term self-renewal an pluripotency of human ES cells, presumably by inhibiting BMP induced extraembryonic lineage differentiation[22-25].
Although MEFs feeder layers were initially used to co-culture both mouse and human ES cells, signal factors secreted from MEFs to maintain pluripotency of the two types of ES cells are fundamentally different. Sato et al first discoveried that TGF-β and Nodal genes were highly expressed in undifferentiated human ES cells[26]. Beattie et al later reported that Activin A, a member of the TGF-β superfamily, was secreted by MEFs, and medium enriched with activin A can replace MEF feeder-layers or MEF-conditioned media to maintain human ES cells in an undifferentiated state[27]. In consistence, James et al demonstrated that the TGF-β/Activin/Nodal pathway was activated through the transcription factors Smad2/3 in undifferentiated human ES cells[28]. The notion that TGF-β/Activin/Nodal signaling supports human ES self-renewal and pluripotency is further supported by the fact that recombinant Activin or Nodal stimulation induces higher levels of pluripotent protein expression (Oct4 and Nanog), while inhibition of TGF-β/Activin/Nodal signaling with Lefty or Follistatin decreases expression of these pluripotent proteins in human ES cells[29, 30].
Recent studies have focused on understanding the molecular mechanisms of TGF-β/Activin/Nodal signaling in retaining human ES cells pluripotency. Xu and colleagues showed that TGF-β/Activin/Nodal signaling activated Smad2/3 which subsequently binds to the Nanog promoter in undifferentiated human ES cells to induce expression of Nanog, a pluripotent transcription factor[31]. Additionally, mutating the putative Smad-binding sites reduced the response of Nanog to modulation of TGF-β signaling[31]. Nanog was also shown to coordinate with Smad2 in a negative-feedback loop to inhibit human ES cell differentiation[32]. In contrast to its important role in maintaining human ES cell pluripotency, the TGF-β/Activin/Nodal signaling is not essential for pluripotency of mouse ES cells. Although this pathway was shown to be active in undifferentiated mouse ES cells as assessed by phosphorylation of smad 2/3, inhibition of smad 2/3 phosphorylation by SB431542 had no effect on the undifferentiated state of mouse ES cells[28]. However, the TGF-β/Activin/Nodal signaling may play a role in mouse ES proliferation. A recent study showed that Inhibition of TGF-β/Activin/Nodal signaling by Smad7 or SB-431542 dramatically decreased mouse ES cell proliferation without effect on their pluripotency[33].
GDF-3 is another TGF-beta superfamily member that plays opposite roles in mouse and human ES cells. GDF-3, which acts as a BMP antagonist by direct binding to BMP-4, is specifically expressed in the pluripotent state of both mouse and human ES cells[34]. Ectopic expression of GDF-3 leads to the maintenance of pluripotency in human ES cells, whereas a similar effect is observed in mouse ES cells when GDF-3 levels are decreased. In the absence of LIF, GDF-3-deficient mouse ES cells can still sustain pluripotent markers[34]. These results are consistent with previously discussed BMP signals which can promote pluripotency of mouse ES cells, but cause differentiation of human ES cells. Thus lower concentrations of BMP antagonists, such as GDF-3, may enhance pluripotency in mouse ES cells, whereas higher levels of GDF-3 may favor pluripotency of human ES cells by abrogating BMP signaling.
The importance of Fibroblast growth factor (FGF) signaling for human ES cells pluripotency is highlighted by the facts that human ES cells are traditionally cultured in the presence of Fibroblast growth factors (FGFs) either on fibroblast feeder layers or in fibroblast-conditioned medium[35, 36]. Studies have demonstrated that all four FGF receptors (FGFR1, FGFR3 and FGFR4) and several components (SOS1, PTPN11 and RAF1) of their downstream activation cascade are significantly upregulated in undifferentiated human ES cells, in comparison to differentiated human ES cells[37-39]. In consistence, withdrawal of FGFs or inhibition of FGF signaling by a FGFR inhibitor, SU5402, rapidly induces human ES cell differentiation[40-42].
Although the pluirpotency maintenance role of exogenous FGFs in human ES cell has been known for a long time, the molecular mechanisms by which they function remain unclear. FGFs signal by binding to FGF receptors (FGFRs), and activate multiple signaling cascades, including Mitogen-Activated Protein Kinases (MAPKs), the Janus kinase/signal transducer and activator of transcription (Jak/Stat), phosphatidylinositol 3-kinase (PI3K) and phosphoinositide phospholipase C (PLCg) pathway[43]. Several studies have highlighted the FGF contribution to the maintenance of human ES cells mainly through the FGF/MEK pathway (Figure 1), [44, 45]. Studies have showed that FGF2 induces feeder layer cells to secret TGFβ1 and insulin-like growth factor 2 (IGF2), which can subsequently promote the undifferentiated state of human ES cells[46, 47]. Bendall et al further reported that the function of exogenous FGFs in promoting ES self-renewal could be replaced by addition of IGF2 alone, suggesting an indirect role of FGFs for human ES cell growth. However, this model was challenged in subsequent publications from Wang et al who reported that exogenous IGF2 alone was insufficient to maintain undifferentiated growth of human ES cells, and they proposed that FGFs may play a direct role in blocking caspase-activated apoptosis through anoikis in human ES cells[48]. Recently, Eiselleova and colleagues postulated a new model whereby endogenous FGF-2 signaling maintained the undifferentiated state and survival of human ESCs, while exogenous FGF-2 mainly suppress cell death and apoptosis genes, thus indirectly contributing to the maintenance of human ES cell pluripotency[49].
FGF signaling in mouse ES cells has also been extensively investigated. Mouse ES cells genetically deficient in Fgf4 and extracellular-signal regulated kinase 2 (Erk2) differentiate inefficiently. These results can be reproduced using inhibitors of FGF receptor and ERK, suggesting blockage of the FGF/MEK signaling pathway promotes mouse ES cell pluripotency[50-52]. Indeed, serum-free mouse ES cell medium supplemented with FGF/MEK inhibitors and LIF permits the derivation of mouse ES cells in the absence of feeders from strains normally considered non-permissive[53]. In addition, a recently identified compound, Pluripotin/SC1, has been shown to maintain mouse ES pluripotency by inhibiting ERK1 and activating the phophoinositide-3 kinase (PI3K) pathway through blocking RasGAP[54-56] [57, 58]. Although inhibition of FGF/MEK pathway can attenuate ES cell differentiation, it is insufficient to support mouse ES cell self-renewal. Combination of the MEK inhibitor PD0325901 with the Glycogen synthase kinase-3 (GSK-3) inhibitor CHIR99021 (known as 2i) can efficiently sustain the pluripotency of mouse ES cells in the absence of exogenous cytokines[59, 60]. Several groups demonstrated that improvement of mouse ES cell pluripotency by inhibition of GSK-3 occurred via Wnt/β-catenin signaling, whereas many others argued that GSK3 was likely to exert β-catenin independent effects in ES cells[59, 61-67].
As demonstrated above, human and mouse ES cells are both derived from blastocyst-stage embryos, but they require different biological signals for maintaining pluripotency. In general, mouse ES cells maintain their pluripotency by activating LIF/STAT3 and BMP signaling, while human ES cells require TGF-β/Nodal and FGF/MEK pathways. Interestingly, several pathways, such as BMP and FGF/MEK, have completely oppositing effects on maintaining the pluriotency of mouse and human ES cells. Activation of BMP signaling and inhibition of the FGF/MEK pathway promote mouse ES self-renewal, whereas inhibition of BMP signaling and activation of FGF/MEK pathway sustain human ES cell pluripotency. These distinct signaling effects on pluripotency may reflect intrinsic differences between mouse and human ES cells. Recent studies have demonstrated that conventional human ES cells do not represent the “ground or naïve state” of stemness, but rather a more developmentally mature “primed state” resembling mouse epiblast stem cells (mEpiSCs) found in the post-implantation, pre-gastrulation stage of embryos [68-74]. Conventional human ES cells exhibit numerous similarities to the mouse EpiSCs over mouse ES cells (Table 1). For instance, conventional human ES cells and mouse EpiSCs display flattened cell colonies and epigenetic X-chromosome inactivation (XiXa), and require Activin and FGF for pluripotency maintanince. In contrast, mouse ES cells exhibit dome-shaped colony morphology and epigenetic activation of both X-chromosome (XaXa), and require LIF/STAT3 signaling to promote self-renewal. Subsequent studies have demonstrated that the medium containing “2i” (MEK inhibitor and GSK-3 inhibitor), when supplemented with other factors (such as forskolin), can efficiently convert conventional human ES cells into a ground or “naïve” state with display of hallmark features of mouse ES cells. This medium can also maintain human ES cell pluriptoency at the naïve state [69, 70, 72, 75-78].
\n\t\t\t\t | \n\t\t
Comparison of the properties of mouse ES cells (mESCs), mouse epiblast stem cells (mEpiSCs), human ES cells (hESCs) and human iPS cells (hiPSCs).
ES cell pluripotency is conferred by a unique transcriptional network[79]. Early global transcriptional profiles and genetic studies have identified several critical transcription factors that are required for the pluripotency of ES cells, such as Oct4, Sox2, Nanog, Foxd3 and Id, etc [80-88]. Here we will mainly focus on Oct4, Sox2 and Nanog, three key transcription factors of the core pluripotency transcriptional network.
OCT4 (also known as Oct3), a POU domain-containing transcription factor, was one of the first transcription factors identified as essential for both early embryo development and pluripotency maintenance in ES cells[84, 89]. The expression of Oct4 is activated at the 8-cell stage and is later restricted to the inner cell mass (ICM) and germ cells in early mouse embryogenesis in vivo [89-92]. Oct4 is highly expressed in both human and mouse ES cells, and its expression diminishes when these cells differentiate and lose pluripotency. Oct4 regulates a broad range of target genes including Fgf4, Utf1, Opn, Rex1/ Zfp42, Fbx15, Sox2 and Cdx2[93-95]. Repression of Oct4 activity in ES cells upregulates Cdx2 expression, leading to ES cell differentiation into trophectoderm[96]. Oct4 is also known to activate downstream genes by binding to enhancers carrying the octamer–sox motif (Oct–Sox enhancer), for synergistic activation with Sox2. In contrast with its target genes, little is known about Oct4 upstream regulators. The Oct4 promoter contains conserved distal and proximal enhancers that can either repress or activate its expression depending on the binding factors occupying these sites[97, 98]. The precise level of Oct4 is important for ES cell fate determination. Loss of Oct4 causes inappropriate differentiation of ES cells into trophectoderm, whereas overexpression of Oct4 results in differentiation into primitive endoderm and mesoderm[99, 100].
Sox2 is an HMG-box transcription factor that is detected in pluripotent cell lineages and the nervous system[101-103]. Inactivate Sox2 in vivo results in early embryonic lethality due to the failure of ICM maintenance[102]. Sox2 can form a complex with the Oct4 protein to occupy Oct–Sox enhancers to regulate target gene expression. Oct–Sox enhancers are found in the regulatory region of most of the genes that are specifically expressed in pluripotent stem cells, such as Oct4, Sox2, Nanog, Utf1, Lefty, Fgf4 and Fbx15[93, 94, 104-108].
Nanog is another homeobox-containing transcription factor that is specifically expressed in pluripotent ES cells. The essential role of Nanog in maintaining the pluripotency of ES cells is highlighted by the facts that Nanog-deficient ES cells are prone to differentiation, whereas forced expression of Nanog partially renders ES cells self-renewal potential in the absence of LIF[85, 86, 109]. How Nanog regulates stem cell pluripotency remains entirely unknown. Studies have indicated that Nanog may maintain ES cell pluripotency by 1) downregulating downstream genes essential for cell differentiation such as Gata4 and Gata6 and 2) activating the expression of genes necessary for self-renewal such as Rex1 and Id[19, 85, 86]. Although it is widely accepted that Nanog, like Oct4 and Sox2, play a central role in pluripotency maintenance, this dogma has been challenged by a subsequent report that Nanog protein levels are undetectable in a fraction of ES cells that express Oct4, and the pure populations of Nanog−/− ES cells can be propagated without losing expression of other pluripotency markers[110].
Little is known about the mechanism by which Nanog is regulated in ES cells. Recently, Suzuki et al showed that Nanog expression was upregulated by BrachyuryT and STAT3 in mouse ES cells[111]. In human ES cells and in mouse EpiSCs, Vallier et al reported that Activin/Nodal signaling stimulated expression of Nanog, which in turn prevents FGF-induced neuroectoderm differentiation [112]. In addition, several studies indicated that the Oct4/Sox2 complex was directly bound to the Nanog promoter to regulate target gene expression [106, 107, 113]. Genomic studies have revealed that Oct4, Sox2, and Nanog frequently bind the same regulatory regions in undifferentiated mouse and human ESCs, and that these binding sites are often in close proximity to one another[113-116]. These results indicate that Oct4, Sox2, and Nanog may physically interact with each other and coordinately regulate target genes in some cases. Additionally, Goke and colleagues reported that combinatorial binding sites of the Oct4/Sox2/Nanog were more conserved between mouse and human ES cells than individual binding sites were [113, 114, 117-119].
Understanding the molecular mechanism of pluripotency can greatly expand our knowledge of ES cell biology and facilitate future stem cell clinical applications. In the past few years, we have seen tremendous advances in understanding ES cell pluripotency. Although mouse ES cells and conventional human ES cells require distinct signaling pathways to maintain pluripotency, they display similar gene expression profiles, activities of transcription factors (such as Oct4, Nanog and Sox2) and transcription factor networks. Our understanding of pluripotency has been further expanded by the advent of iPS cells and the very recent discovery that conventional human ES cells are more equivalent to mouse EpiSCs, but rather “naïve state” of mouse ES cells. Nevertheless, our knowledge of the molecular mechanisms of ES cell pluripotency is still very limited. For instance, it remains unknown how growth factors establish and control transcriptional networks to regulate pluripoency and how ES cells respond so precisely to exogenous cues. Given the rapid advance in ES cell biology, we anticipate the molecular mechanisms underlying pluripotency of ES cells will soon be uncovered and pluripotent stem cells, such as ES cells and iPS cells, will be widely used for clinical applications in the near future.
This work is funded by the seed fund of the Western University of Health Sciences.
3D printing is an additive manufacturing (AM) process that enables the manufacturing of components with complex geometries in a layer-by-layer fashion. 3D printing became popular after the first machine was introduced to the market in 1986 by Hull [1]. Charles Hull created the first stereolithography (SLA) manufacturing method which he used for the rapid design and manufacturing of small prototype plastic parts. Stereolithography uses light to activate polymers within a resin (photopolymerization) to create 3D, complex shapes [2, 3]. This SLA system was commercialized in 1987 by the company 3D Systems. Since this breakthrough invention, there has been great effort in producing machines that can process a variety of plastics. Some of the machines currently in the market are fused deposition modeling (FDM) [4, 5] and direct ink write (DIW) for extrusion-based processes [6, 7]. Powder bed fusion (PBF) and laser sintering (SLS) are used for processes requiring a laser to cure or fuse polymeric materials [8]. Inkjet printers also use light to photopolymerize ink drops into complex shapes [9]. Extensive reviews on these processing and 3D printing technologies have been published elsewhere [4, 5, 10, 11, 12, 13, 14]. This chapter focuses on applications that use AM for the 3D printing of polymeric materials.
\nSince the 1980s, 3D printing has become very popular as a result of the rapid manufacturing of components with architectures designed to meet specific applications. AM allows for the manufacturing of a variety of shapes in a layer-by-layer fashion, often without the need of post-processing such as machining. As a general scheme, AM starts with the design of a virtual object using CAD (computer-aided design) software that generates a STL (stereolithography, named after Charles Hull’s SLA process) file format [15]. A slicer program interprets the STL file and converts it into g-code (e.g. Slic3r, 3DPrinterOS, MakerBot Print, and others). The computer controls the stage and dispenser of the 3D printer allowing prototypes to be manufactured. Rapid prototyping allows one to refine product ideas while saving significant time and money because it allows for iterations prior to creating a final product. Optimization via an iterative process involves touching and feeling the prototype, in real time, in order to finalize the shape and geometry, leading to a final product. Characterization methods during iterations and on the final design include optical microscopy, SEM, and mechanical tests. Others methods, such as bio-compatibility (cell-adhesion and proliferation) and electrical performance are performed depending on the application. Figure 1 demonstrates a general scheme for the AM process. Despite the many advances in AM, the technology still has many challenges that need to be addressed. These challenges are related to the speed of the processes (which in many cases is slower than injection molding processes and machining), cost of the machines, and limited feedstock. However, advantages outweigh the challenges due to the fact that AM allows for compositional flexibility, complex macro and microstructures, and easy modeling and optimization. As a result, industries including biomedical engineering, transportation, and the military have adopted AM as the main manufacturing method for the printing of prototypes and final parts [16, 17].
\nGeneral scheme for the use of additive manufacturing processes, from the choice of material to the final product. The 3D printing of parts involves the use of a computer-assisted design software that generates a STL file format that is then sliced and formatted into gcode. The computer controls the stage and dispenser to generate materials with specific architectures, e.g. faced-centered tetragonal cushion using direct ink writing (a) and diamond structure using FDM (b).
Careful attention is imperative when choosing a material to print a given part. While there are a variety of commercially available polymers, not one polymer is inclusive and will give one the properties needed for a specific application. Furthermore, a single AM technique is not capable of printing any one individual polymer available in the market. The selection of material depends on the application and the customers’ needs. Figure 2 lists the decision criteria for the selection of a material. One must take into consideration the environment at which the part will be exposed and the properties required (e.g. temperature, mechanical load, humidity, chemical exposure, radiation, UV light), the processability, 3D printing method, and availability.
\nMaterial selection chart for product design and manufacturing.
Polymers have become consumer goods, for they are used to manufacture bottles, toys, tools, bags, phones, computers, tools, cushions, electronics and transportation components [18]. Thus, it makes sense that efforts have focused on developing materials that can be 3D printed, which allows for rapid manufacturing [2, 3, 4, 17]. Table 1 lists commercially available polymers used in some of the AM processes. Polycarbonate (PC), acrylonitrile butadiene styrene (ABS), poly ether ester ketone (PEEK), polyetherimide (ULTEM) and Nylon are common polymers used in processes requiring thermoplastics, or plastics that are processed by heating to a semi-liquid state and close to the melting point. Upon extrusion, the printed layers fuse and solidify. AM techniques that use thermoplastics are Fused-Deposition Modeling (FDM), Jetting (InkJet), and Selective Laser Sintering (SLS). SLA and Direct Ink Writing (DIW) use thermosetting polymers in their liquid state, or polymers that become solids after curing. A chemical reaction occurs prior to the melting point, resulting in a solid-state material. In SLA and DIW, polymers are formulated to meet specific properties, most importantly rheological. For example, each layer should be self-supporting and should allow for the printing of multiple layers while retaining the designed geometry [14, 19, 20, 21]. Rheologically, this corresponds to a resin that has a yield stress at high oscillatory stresses, such that the resin is solid-like at rest (low stress) and liquid like during flow (high stress) [7]. One of the main challenges in the polymer 3D printing industry is the limited feedstock available for purchase. Polymers listed in Table 1 cannot be used in all applications. Particularly, polymers in the pure state lack mechanical strength for load-bearing applications. The addition of fillers, such as silica [22, 23] and carbon fibers [24, 25], is often used to generate materials with high mechanical strength. Furthermore, the incorporation of additives enhances materials properties by adding functionality to the parts that include getter [20], UV and radiation resistance [26, 27, 28], and anti-fouling properties [29, 30, 31].
\nAM technology | \nProcess | \nPhysical state of starting material | \nFeedstock | \n
---|---|---|---|
FDM | \nMelting-solidifying | \nSolid | \nPC, ABS, PLA, ULTEM, Nylon, Carbon-filled Nylon, ASA | \n
SLA | \nPhotocuring | \nLiquid | \nThermosetting- acrylates and epoxy | \n
SLS | \nMelting-solidifying | \nSolid | \nPCL, PLA | \n
Jetting | \nPhotocuring | \nSolid | \nABS, ASA, PCL, PLA, Vero | \n
Direct Writing | \nExtrusion-heat/UV curing | \nliquid | \nThermosetting- any material with adequate viscosity | \n
List of polymers used for 3D printing applications.
The biomedical market represents 11% of the total AM market share today, and will be a strong driver for AM development and growth [32]. Since the early 2000s, there has been increased interest in using 3D printing to fabricate hard tissues (bones, teeth, cartilage) and soft tissues (organs, skin, and others) [2, 3, 4, 16, 33]. The manufacturing of prostheses and scaffolds with complex geometries is especially important for regenerative medicine, where a porous scaffold is implanted into the patient to serve as a template for tissue to regenerate while the implant degrades slowly in the body. Other implants need to stay in place for the lifetime of the patient. 3D printing allows for the rapid manufacturing of customized prosthetics and implants with controlled architectures. The structure can be designed through the translation of x-ray, MRI, and CT images into STL file formats. The STL file can be processed by software and a design can be generated based on the patient’s specific needs. Metals are commonly used to generate prosthetics for bone reconstruction. ABS and PLA are the most suitable non-biodegradable polymers used for the manufacturing of scaffolds. However, materials used in medicine must enable cell adhesion, growth, and differentiation. Current feedstock for biomaterials is limited to collagen, gelatin, fibrin, and chitosan, which are similar to natural tissue, have high affinity to cells and are highly hydrated. The main challenge with these soft natural polymers is their low mechanical strength [33]. In biomedical engineering, the main focus has been on the development of biopolymeric materials for tissue and scaffold generations with improved flexibility, strength, and patient compatibility in order to prevent implant rejection and toxicity. Some polymeric mixtures include living cells isolated from the patient and grown in the laboratory. These types of polymers are often hydrogels suitable for ink jet 3D printing technologies. Table 2 shows various polymers used for biomedical applications. Some examples of biomedical devices developed using 3D printing are implants, prosthetics, dental, orthodontics, hearing aids, and drug release tissues.
\nMaterial | \n3D printing techniques | \nComments | \n
---|---|---|
PLA, PCLA, PLGA | \nFDM | \nScaffolds. Biodegradable. Can add fillers, e.g. HA, for improved cell adhesion and mechanical properties | \n
Collagen, alginate, PEG, fibrin, chitosan | \nInkjet, extrusion | \nBiodegradable scaffolds. Can add fillers and cells for improved cell adhesion and mechanical properties | \n
PCL, methacrylate copolymers | \nSLS | \nBiodegradable scaffolds. Improved mechanical properties | \n
Polymers and processes used for the additive manufacturing of biomedical devices.
Polymers used for tissue and organ fabrication need to have various functions in order to (1) allow for cell attachment and migration, (2) transfer growth factors and waste products, (3) maintain its shape while cells are growing and (4) maintain adequate mechanical properties. Wu et al. [34] reported the generation of a biopolymeric material based on chitosan dissolved in an acid mixture of acetic acid, lactic acid, and citric acid. This biomaterial was 3D printed using an ink-writing technique, then dried under vacuum and neutralized to remove any acid residue. The structure of the scaffold was characterized using confocal laser scanning microscopy and the images showed wrinkles attributed to the volume change. Tensile mechanical tests show that the printed material exhibits a strain to failure of 400% under tensile load and a 7.5 MPa ultimate strength when in its neutralized form. Furthermore, the 3D printed material allows for excellent cell adhesion, growth, and proliferation, as demonstrated using the Live-Dead staining method, fluorescence microscopy, and SEM.
\nLuo et al. [35] reported the 3D printing of a bioceramic hollow struts-packed scaffold using an extrusion typ. 3D printer and a shell/core nozzle. The ink contained Ca7Si2P2O16, alginate and Pluronic F-127. After printing, the ink was dried overnight and sintered for 3 hours at 1400°C to remove the alginate and F-127 materials. The morphology was analyzed using an optical microscope. The micropores and the microstructure of the pores were characterized using SEM. The fabricated scaffolds (16/23 shell/core size) were subjected to mechanical testing and exhibited a compressive strength of 5 MPa, comparable to cancellous bone (2–12 MPa), and a modulus of 160 MPa. The scaffold had high porosity (65–85%), adjusted with the core/shell size nozzles. The high porosity and surface area (up to 6500 mm2/g) allowed for cell adhesion and proliferation on the outer and inner surface of the scaffold, as determined by SEM. Finally, the in-vivo bone formation study in a rabbit demonstrated that the bioceramic implant allows for good cell integration and bone formation was detected with micro-CT.
\nLewis’ team at Harvard University 3D printed a tympanic membrane scaffold composed of PDMS, PLA, and PCL based materials using a DIW technique [36]. The team demonstrated that it is possible to design and fabricate materials with similar properties when compared to human specimens. The high frequency displacement and acoustics were organized by concentric rings for each 3D printed graft, and it was very dependent on the patterns and mechanical properties, characterized via digital opto-electronic holography, laser Doppler vibrometry, and dynamic mechanical analysis. In a different study, the team 3D printed cellular materials with vascular networks for flow [37]. The 3D printed structure was fabricated using an ink composed of Pluronic F-127, GelMA (gelatin methacrylate to allow for UV curing) and fibroblast cell culture. After curing, the Pluronic F-127 was removed by cooling to 4°C, yielding open channels that represent the vascular networks. Lewis’ team demonstrated that blood and other cellular liquids can flow through the channels with minimal death of cells.
\nPatients with skin burns and thick wound injuries often suffer from long term recovery and extensive and expensive treatments. The autologous split-thickness skin graft (ASSG) is the technique most often used to treat large wounds [38]. A skin tissue is place in the injured area and assists with the wound closure and healing. This technique relies on the removal of a piece of skin from a different part of the patient’s body and reapplying it on the place of injury. The drawback with ASSG is that it is limited by the size of donor sites and also creates another place of injury [38]. 3D printing of biomaterials would alleviate the problems related to ASSG. Skin cells are cultured in a laboratory and mixed with biocompatible polymers for bioprinting. In 2012, Koch Singh et al. [39] reported the 3D printing of skin using a laser-based inkjet printing method. The inks were composed of blood plasma/alginate solution and fibroblast/keratinocytes/collagen biomaterials. Collagen is the main component of the extracellular matrix (ECM) in skin. The team proved that the laser-based printing method does not harm the cells by performing proliferation of the cells in histologic sections 10 days after printing. Ki-67 staining, which includes the protein present in cells during their active cell cycle phases, shows that proliferating cells can be found in all regions, verifying vitality. In addition, a build-up of basal lamina, cell adhesion and proliferation- sign of tissue generation was observed.
\nThe dental industry is taking advantage of 3D printing technologies for restoratives, implants, and orthodontics purposes. Currently, professionals in the dental field have access to 3D printers and it is possible to print designs in a clinical environment. A CT scan is used to generate a defined shape based on the patient’s morphology and quickly fabricate and replace a missing tooth [40]. 3D printing is used for the manufacturing of aligners, braces, dental implants, and crowns [40]. Biocompatible materials are used for the fabrication of dental parts using 3D printing, e.g. polylactic acid, polycaprolactone and polyglycolide, and acrylates [3]. It is possible to fabricate dental implants with antibacterial properties by the incorporation of additives, such as quaternary ammonium salts [41, 42, 43]. At the age of 23, Amos Dudley fabricated his own orthodontic aligners while he was a student at New Jersey Institute of Technology [44]. He used equipment available at the institute to scan and print models of his teeth. A non-toxic plastic was used to mold and eventually generate 12 clear aligners. Amos had access to a Stratasys Dimension 1200 3D printer and used a mixture of alginate powder and PermaStone as the resin to print the aligners, which were tested by fitting them on his teeth. While it was not a trivial problem to solve, Amos proved the ability of 3D printing orthodontic materials for teeth alignment.
\nAM has been widely used in the biomedical industry and will continue to impact work in the future. Some challenges will persist, such as regulatory issues, limited materials, and inconsistent quality [45]. AM biomedical products require FDA approval, which can be time consuming and difficult to obtain [46]. Biocompatibility will require the development of new techniques and materials to produce high quality, high performing AM materials [47]. Furthermore, mechanical properties of AM materials need to be well assessed such that final properties can have reliable and reproducible behaviors. Further development for on-demand and patient-specific applications will be exciting work in this field. For example, designing patient-specific implants following a CT-scan will result in quick results [48]. Complex parts with specific mechanical properties and biocompatibility can be constructed on demand and with multifunctional components if needed. AM Research and development may help to improve bio-printed scaffolds and tissues for clinical applications to reduce cost for tissue engineering [49]. Manufacturing AM artificial organs, which includes multifunctionality (i.e. bionic ear [50]), will revolutionize the field of 3D printing for biomedical applications.
\nOne of the most promising fields in the future of AM is the aerospace industry. According to Wohlers’ report, this industry account for almost 20% of the total AM market today [32]. Aerospace applications typically require light weight and high strength materials. The importance of AM relies on the reduced cost, increased flexibility of design, and increase in a variety of products to meet customer needs. Additive manufacturing is an important technology that enables the design and manufacturing of complex structured products with improved mechanical strength and lower weight, at a lower cost and reduced lead-time. The aerospace industry has replaced the conventional manufacturing methods of molding and machining with 3D printing technology for small scale production. At a small production scale, AM offers effectively low-cost design and assembly [17].
\nThe aerospace industry implemented the use of AM approximately 20 years ago [51]. The main use for 3D printing has been focused on prototyping, modeling and producing jigs, fixtures and tools [17]. Furthermore, AM is used to build replacement parts on-demand when required. The ability to build on-demand spare components reduces costs for the production of parts that may never be used due to them becoming obsolete to new technology, which also saves warehouse storage space. For example, BAE Systems is currently 3D printing window breather pipes used in jetliners [52]. These pipes cost 40% less than pipes manufactured using injection molding processes and are manufactured on an as-needed basis.
\nRecently, NASA designed a rover, named Desert RATS, that can support humans in a pressurized cabin in space [53]. The rover is intended to transport humans to Mars. It contains 70 3D printed parts that include flame-retardant vents and housings, camera mounts, large pod doors, front bumpers, complex electronics, and others. The materials used for the 3D printing of the part used in the rover were ABS, PCABS and PC, and were printed using a FDM Stratasys 3D printer. Piper Aircraft manufactures tools using PC that can withstand hydroforming pressures of 3000 to 6000 psi. Aurora Flight Science additively manufactured wings that weigh one third of the fully dense metal components [54]. Some wings have integrated electronics. Lepron generated 200 different designs for use in piloted helicopters [17]. It is foreseen that aerospace companies will replace small components with 3D printed parts, thus reducing the weight of the machines. Some examples are arm rests, seat belts, food trays, and many others [17].
\nCompanies have adopted AM for fast production without making substantial changes to their products [17]. This modification is mostly due to the fast-changing market and low cost of generating such small builds. Several challenges would have to be overcome to facilitate the growth of AM. Some of these challenges include: (1) current speed of AM machines is slow for bulk production; (2) few polymeric material options; and (3) current machines do not allow for the manufacturing of large components [17, 55]. In the future, it is expected that companies will pursue a completely different business model by performing product customization for end-product while maintaining the on-demand part supply. Future work will focus on the development of multifunctional structures with complex geometries, which allows for novel solutions for complicated problems. AM techniques, such as using functionally graded materials, can be used in order to tailor the mechanical and/or thermal response of components [56]. Furthermore, on-demand manufacturing will reduce costs and eliminates potential damage caused by storage [45].
\nElectronic devices require suitable mechanical, geometrical, and optical functionalities to allow for miniaturization, low energy consumption, and smart capabilities [57]. The production of prototypes and end-products has to rapidly change due to the fast-changing technology. The conventional method for manufacturing electronic devices is using subtractive methods that involve masking and etching of sacrificial materials [58]. AM allows for the reduction of material waste, energy consumption and processing time and steps. 3D printing is being used to substitute steps for mounting and assembling electronic devices [59]. The additive process deposits material in a controlled layer-by-layer process allowing the manufacturing of complex geometries and dimensions. In addition, it enables 3D orientation of important components to improve performance. With miniaturization, AM allows for the manufacturing of small parts that would otherwise be difficult to obtain. AM has found application for thin films [60], inductors [61], solar cells [62], and others. The most common 3D printing techniques for electronics are inkjet and direct writing of conductive inks.
\nJennifer Lewis and colleagues fully 3D printed a quantum-dot (QD) light-emitting diode (LED) system, including green and orange-red light emitters embedded in a silicone matrix [63]. The printed device exhibits a performance of 10–100-fold below the best processed QD-LEP but could potentially be optimized with the addition of an electron-transport layer. A copper nanoparticle stabilized with polyvinyl pyrrolidine was mixed with 2-(2-butoxyethoxy)ethanol to prepare ink for inkjet printing [64]. The ink was printed onto a polyimide subtracted and sintered at 200°C. The prepared electronic device resulted in low electrical resistivity (≥ 3.6 μΩcm, or ≥ 2.2 times the resistivity of bulk copper). Bionic ears were printed using an inkjet printer [50]. The inks were composed of cell-cultured alginate and chondrocytes hydrogel matrix and a conductive polymer consisting of silicone and silver nanoparticles. The 3D printed ears exhibit enhanced auditory sensing for radio-frequency reception allowing the ear to listen to stereo music. This result demonstrates that bioengineering and electronics can be merged, resulting in advanced technologies. Students from Northwest Nazaren University and Caldwell High School designed the 3D printed CubeSat [65]. The CubeSat was launched aboard Delta II rocket as part of a NASA mission in 2013. It carries miniaturized electronics and sensors and is intended to collect real-time data on the effects of the harsh environments of space (oxygen, UV, radiation, temperature and collisions) on the polymeric materials- ABS, PLA, Nylon, and PEI/PC ULTEM.
\nFuture research and development in the electronics field will take advantage of low cost methods, flexibility in design, and fast speed of 3D printers for designing and prototyping new products. For example, printing circuit boards will offer superior accuracy and flexibility, with potential cost savings, environmental impacts, faster production times, and increased design versatility. Furthermore, adaptive 3D printing, which takes advantage of a closed-loop method that combines real-time feedback control and DIW of functional materials to construct devices on dynamic surfaces, is an exciting field of research [66]. This method of 3D printing may lead to new forms of smart manufacturing technologies for directly printed wearable devices. New possibilities will emerge in the wearable device industry, in biological and biomedical research, and in the study and treatment of advanced medical treatments.
\nUnsurprisingly, the amount of plastic pollution on the planet is alarming [67]. Plastics have dominated our marketplace due to their utility and versatility and make up at least 10% by mass of our waste streams. Plastics are designed to be durable and to withstand harsh environmental conditions. Therefore, the amount of plastic waste is only expected to increase in the future. Currently, 91% of plastic is not being recycled. The negative impact plastics have on our ecosystem is well recognized and researchers are using this as a business model and opportunity [68, 69]. Considerable efforts are being placed on recycling and reusing plastic waste. Prof. Sahajwalla at the University of New South Wales Sydney and her team work on turning plastic waste into usable polymers, including 3D printing polymers [70]. The company Reflow is collecting polyethylene terephthalate (PET) waste bottles and turning them into filaments suitable for 3D FDM printers [71]. A company in Belgium, Yuma, is using recycled plastics for the 3D printing of sunglasses [72]. The U.S. Army Research Laboratory and the U.S. Marine Corps are working together to repurpose plastic waste by printing items from recycled plastic useful for soldiers [73]. This process allows for a decrease in transportation costs and manufacturing of parts on demand. This large effort is expected to have a positive impact on both the environment and communities by turning polymer 3D printing into income for waste collectors and removing waste from the streams.
\nIndustries are moving toward the implementation of 3D printing as a manufacturing process because it facilitates the design of complex structures and rapid production of prototypes. AM utilizes a computer-aided design software that allows for the design of architectures with defined porosity and structures at a microscopic level. Because of the easy production of 3D printed prototypes, modeling based on a specific application can be performed to further improve the design of the end product and potentially reduce failure risks. The 3D printing of polymers and polymer composites has significantly progressed over the last 40 years and is expected to increase in the near future. Thermoplastic materials are readily commercially available for use in FDM, SLS, and inkjet processes. Materials like PC, ABS, PLA, ULTEM, and PCLA are commonly used for the manufacturing of tools, prototypes, and items used in the aerospace industry. However, these polymers are not one-size-fits-all types of polymers and are not necessary a good choice for all applications. Thus, research efforts are focused on developing materials that are capable of meeting specific applications. For examples, polymers blended with cultured cells can be used for scaffolds and implants on biological systems. Cells can be obtained from the patient and cultivated in the laboratory, thus producing a material that is less likely to be rejected by the patient. Fillers and additives can be used to generate multifunctional materials with improved mechanical properties. Fillers, such as CNTs and graphene, can be incorporated into the polymer to produce a material that is electrically conductive.
\nDespite all of the advances in the design and development of new polymeric materials for AM applications, challenges still remain. The availability of polymeric inks suitable for extreme applications, such as low temperature environments, high load pressures, and radiation resistance, is very limited. The development of new materials is necessary to increase the usefulness of polymer 3D printing technologies. Ideally, some of these composites are recyclable and/or biodegradable to reduce the negative impact plastics have on our environment.
\nWe thank the US Department of Energy’s National Nuclear Security Administration contract DE-AC-52-06NA25396 for providing financial support.
\nThe authors declare no conflict of interest.
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