Modifications of Graphene Oxide-based composites biomaterial promote human Wharton’s Jelly derived Mesenchymal Stem Cells. Functionalization and characterization

Research Paper (postgraduate), 2017
23 Pages, Grade: 3.00




2. Graphene Oxide

3. Graphene Oxide properties

4. Wharton’s Jelly Derived Mesenchymal Stem Cell

5. GO-based composite functionalization
5.1 Cell biological application
5.2 Biomedical device application
5.3 Biosensor application
5.4 Bio-environment and energy application
5.5 Drug carrier application

6. GO-WJMSC characterization analysis
6.1 Morphology
6.2 Viability
6.3 Phenotypically and purity 13 6.4Multi-potency

7. GO-stem cell future potential applicability

8. Conclusion


Modifications of Graphene Oxide-based composites biomaterial promote human Wharton’s Jelly derived
Mesenchymal Stem Cells: functionalization, and characterization.

Department, Faculty of Sciences and Natural Resources, University of Malaysia Sabah

Y. Umul Hanim *

Abstract- The successful in developing a straightforward preparative route in biomaterial composite fabrication with the great promising Umbilical Cord Matrix derived mesenchymal stem cells (MSCs) known as Wharton’s Jelly also well known as fetal MSC. WJMSC has been created a reliable modernist and briskest innovative research method especially in manipulation of Graphene Oxide (GO) as a new biomaterials loaded with an ample advantages in regard of synthetic materials that applicable for a replacement or regenerating a living system. Bio-interface in mesenchymal stem cell certainly brighten the hope of all stripes scientist researchers in aspiration of biomaterial application whilst can impact the benefit in the fields of biomedical, biotechnology, bioengineering, pharmacology and biosensors yet the finding remain a number of challenges in the fast-growing field. The purpose of this review, we enumerated the key aspect an earmark that will be focused on GO functionalization, and GO-WJMSC characterization, accordingly to GO-WJMSC application in enhanced the proliferation, and multipotency for a brighter future potential of GO-stem cells applicability.

Index Terms- Graphene Oxide, Graphene Oxide-based composite, substrate, Umbilical Cord Matrix Mesenchymal Stem Cells, Wharton’s Jelly, functionalization, viability, multipotency, mesenchymal stem cell application.

1. Introduction

The studies of biomaterial substrates have been accepted without ample of questions for more than a generation (Baker, 2011) since it was discovered in the 1900s (Bergmann and Stumpf, 2013). The great field of biomaterial composite - stem cell technology in future advancement rely on the effective of robust designation throughout privilege materials composite integration. Therefore, the next horizon in the fast-growing field which in biomaterial composites exceptionally remain crucial challenges in elucidating the complexity understanding in advance modification of new germinated biomaterial namely graphene oxide (GO) loaded with complete advantages package particularly will enumerated in this review with the meaning of synthetic materials applicability in the promoting the stem cell behavior for instances in multipotency of stem cell (Lee et al., 2011: Nakajima et al., 2007:Luo et al., 2015:Kim et al., 2013) precisely serve as a transferable and implantable application evenly giving high impact of benefit in the fields of biomedical, biotechnology, bioengineering, pharmacology, biosensors (Park et al. 2015), energy conversion and storage (Li et al., 2015). Alternatively, exceptional innovative in GO composite attained a numerous attempting among scientist recently. Regardless in variety fields lured by the researchers, ideally the phase of clinical trial of biomaterials-human Mesenchymal Stem Cells (hMSCs) still infancy stage even the discovery of GO .

MSCs therapy relies on a larger-scale of derivative sources in the various preparations to biomedical application as revealed in tissue engineering, drug delivery, gene delivery and bioimaging (Rahul et al. 2016). Study from Zhao et al. 2015 dictated that MSCs can be isolated from umbilical cord, namely Wharton’s Jelly up to a point of other sources as surely from bone marrow, placenta, fat, amniotic fluid (Kim et al. 2015) and also from urinary membrane (Bharadwaj et al. 2011) and enable to comply in 3-dimentional, 3D innovation without restricted regardless stem cell behaviors. WJMSCs attained a workable source basically in tissue engineering approach because it readily isolated, expended, cell direct injection and transplanted either intra-peritoneum or intravenous (Neuss et al., 2008; Nakia et al., 2010:Gu et al., 2014: Zhou et al., 2015).

Graphene Oxide (GO) biomaterial has been ideally proven as a novel material notably interest in the field of biomedical as for in vivo implantation application which vast in number of orthopedic therapy significantly (Garcia et al. 2016) in a position of its excellent amphiphilic compound properties specifically synthesis in a heavily oxidized by oxidation and exfoliation procedures of graphite (Liu et al. 2013; Rosa et al. 2016 ; Rahul et al. 2016; Park et al.2015; Lee et al. 2011:Gu et al., 2014), which is derived from the two-dimensional carbon from carboxyl group, epoxide and hydroxyl group, draw amazing effect to the physical, mechanical and biological properties of cell lines enable interaction by amphiphilic bonding (Shen et al. 2012:Li et al., 2015:Kim et al., 2013: Yang et al., 2009) illustrated in figure 1.

Abbildung in dieser Leseprobe nicht enthalten

As distinct study by Yang et al. 2016, stem cell engineering exploited using chemically exfoliations GO of single or multilayer atomic sheets as highly potential biomedical application due to physicochemical stability, high electrical conductivity, tunable amphiphilicity (Liu et al. 2013: Kim et al., 2015: Saravanan et al., 2017: Li et al., 2012: Dubey et al., 2015), mechanical strength (Yang et al. 2016: Kim et al., 2013) and good biocompatibility evenly allow control of cellular behaviors. The package of these unique properties of GO recently has driven the cellular behaviors for instance cell adhesion, proliferation, differentiation, polarization, and biocompatibility in MSCs on GO substrates or scaffold with regard to remain eliminated in severely declining sternness of adult and fetal derived human-MSCs during in vitro expansion on tissue culture polystyrene (TCPS) presently limits their therapeutic efficacy prior to cell transplantation (Lucas et a. 2016:Gu et al., 2014) by manipulation of natural and synthetic biomaterials innovation, therefore, various attempting in incorporating interface of GO-based biomaterial composite modification have been crystalized to envision the novel finding of efficacy in stem cell behavior. Undoubtedly, the purpose of this review herein to be enumerated the key aspect issues that will be projected on GO functionalization, GO-WJMSC characterization, and GO-WJMSC application in enhanced the MSCs proliferation, and multipotency also future potential of GO-stem cells towards applicability.

2. Graphene Oxide

Since early 2004, GO has effectively modified the process synthesis from the current fabrication including production using mechanical or thermal exfoliation, chemical exfoliation of graphite (Rosa et al., 2016: Sharma et al., 2016), chemical vapour deposition (CVP) and epitaxial growth (Gu et al., 2014). The oxidation of graphite to be graphite oxide is the threshold to fabricate the most effectively and friendly fabrication of GO by employed the mechanical exfoliation in water using sonication purposely to produce a single or few layers of GO whilst increase dramatically as highly desired by scientist due to a cheaper, simpler, more efficient, better quantity and quality compared to present method to be suitable financially for industrial and commercial larger purpose. GO also known as a flakes material that can be visible in yellowish suspension.

3. Graphene Oxide properties

Basically, the unique features of GO known as flakey material has aforementioned widely towards the several package properties enumerated as electrically insulator, physicochemical stability, mechanical strength, and biological compatible. Particularly, GO consist of three reactive oxygen functional group (ROFG) have lifter the properties of electrical insulator inclusively the epoxide, carboxyl and hydroxyl group. Electrically, GO plays a great insulating material due to the practicable of saturated sp3 orbital coordinate the missing carbon atom and negatively charged density species bound to carbon engendered GO as a super capacitor material (Li et al., 2015: Becerril et al., 2008). Schematic is illustrated in Figure 2.

The distinct physicochemical of GO inclusively listed as the easily dispersed due to attraction of hydrophilic, hydrophobic and amphiphilicity that encountered GO as a highly soluble and easy dispersible in water and organic solvent due to the ionizable edge -COOH affected by tunable of pH. That property has promises GO to be dispersed especially in physiological media making GO purposely apply in biomedical diligence (Cote et al., 2011: Kim et al., 2010). In addition, the unique properties of GO especially as a carbon based material lead to the elevation of hydrophilicity and large surface areas driven GO potentially utilize in drug delivery (Saravanan et al., 2017: Li et al., 2012: Dubey et al., 2015). Physically GO suspension can be soluble in water by express in dark brown to light yellowish in color regardless to any concentration.

Additionally, GO (Wang et al., 2011: Luo et al., 2015) assigned as a biocompatibility materials that coordinated the cellular behavior succinctly reported by Luo and colleagues, GO-incorporated Poly(lactic-co- glycolic acid) known as PLGA enhanced the cellular metabolism and osteogenic differentiation importantly as a protein adsorption through the capacity of surface interaction. Due to these unique properties, GO is inevitable to be a favor candidate to be delved in the chemical modification in order to create a better adaptable for a lot of application by functionalization depending on desired application. Accordingly to the study by Wang and group stated that GO in low (0.1mg/ml) and mild dose 0.25mg/ml) is activate the cell growth and consider as a high biocompatibility (Wang et al., 2011).

4. Wharton’s Jelly Derived Mesenchymal Stem Cell

Mesenchymal stem cells (MSCs) parentage is from a layer of mesoderm has been proved of the high capacity for self-renewal and great in multi-lineage differentiation into adipocytes, osteoblasts, chondrocytes and smooth muscle cell (Zhou et al., 2015: Pittenger et al., 1999: Wang et al.,2004). MSCs primitively derived from bone marrow also well-known as an adult stem cell discovered comparable to fetal stem cell evenly fetal stem cell efficacy possible to encounter more beneficial than adult stem cell. MSC has been circulated in the meeting agreement of International Society Cellular Therapy (ICST) by Dominici et al. (2006) which as MSC has three minimal criteria to be defined as truly MSC population, firstly MSC must be plastic-adherent when maintained in standard culture conditions. Secondly must capable to express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79 alpha or CD19 and HLA-DR surface molecules. Lastly MSC enable differentiate to osteoblasts, adipocytes and chondrocytes in vitro. Exceptionally, fetal stem cell has studied possibly derived from multiple tissues and organ for instance umbilical cord (Martin-Pendon et al., 2008), placenta (In ‘t Anker et al., 2004), amniotic fluid

(Tsai et al., 2004), and Umbilical cord blood (Jang et al., 2006). A nifty study of human Wharton’s Jelly derived umbilical cord matríc has brighten the field of MSCs with regard to the special advantage of readily available, free ethically, easily harvested, non-invasively pathogenic and enrichment resources (Zhou et al., 2015). Basically, hWJMSCs are concern to exhibit outstanding advantages over other MSC resources especially for cell-based tissue engineering, cell therapy and regenerative medicine (Pittenger et Al., 1999: Zhao et al., 2015). Successfully stem cell therapy has requested billions of hWJMSC in their application however there is an inevitable factor which may lead to the loss of multipotent ability and rely to cell senescence in cell-based therapy or regenerative medicine. Thus, the multitude effort should be designed for the development of a novel finding and technology that to enhance the yields of hWJMSC while maintaining the proliferation potential and multi-potency of the cell.

Abbildung in dieser Leseprobe nicht enthalten

5. GO-based composite functionalization

Along the lines of GO modified functionalization method, strong emphasize in the superior properties in completely package is enumerated for instance as a unique functional group applicable including epoxide, carboxyl and hydroxyl group may lead to serve a larger surface area, mechanical stability, electrostatic interaction capacity and optical properties which promise its great characteristic among other materials ideally expended commitment on GO application as scaffold either substrate including in vast field of biotechnology, biomedical, biosensor and stretched a head upon in energy platform also in lithium batteries and as a super capacitors (Becerril et al. 2008; Li et al. 2015: Sharma et al., 2016).

The most common GO synthesis is manufactured using first principal by Hummers method implied the oxidation of graphite treated with potassium permanganate and sulfuric acid (Gu et al., 2013: Hummers et al., 1958) nonetheless GO has been considered as one of a low-cost material manufacturing which attract the wide world scientist to give full effort on GO investigation (Dubey et al., 2015: Singh etal., 2009: Sharma et al., 2016).

Generally, there are several common on a solid support substrate have been demonstrated in tissue engineering for instances including glass, polymers, gold or silicon, and thin coatings such as poly-L-lysine, agarose or polyacrylamide (Ghaemi et al., 2013) to a slight variation in emphasize the substrate functionalization through dip or spin coating, plasma treatment, surface grafting, self-assembled monolayer (SAM) formation and micro fluidics approaches.

To address the huge amount of literature in composites functionalization innovatively, herein as to generally summarize that there are enumerate of the key factors as to contemplate the efficacy of composites for instance synchronization integrated factor exhibited the advancement effect of cell phenotypes, materials compositions manifestation (Nancy et al. 2016; Singh et al., 2009). Next, for the second factor is the extended homogeneity compositional material interaction whereas reliability control the cell lines behavior as a 3D scaffold, substrate, or implantation devices (Fisher et al. 2010; Nakajima et al. 2006). For the third factor is the sustainability of biocompatible in versatile technique in the cell-material interaction which detail manifested by Neuss and colleagues in 2008 analyze the variation parameter stem cell material combination for tissue engineering. Views of the above points, seem is loaded with the meaning of materials composite, combination and hybrid material purposely.

Deeply entail on the GO composite owing a huge meaningful of the advantage potential along the materials combination for instances such nanoparticles, nanocomposite (Mahmoudi et al., 2016; Ji. et al., 2016; Zuo et al, 2013), nanofibers (Mahmoudi et al. 2016), and polymer scaffold (Kim et al., 2015). Regardless the vast approaches of GO composite innovation namely construct modification in Extracellular Matrix (ECM) component, administrate of growth factors (Nakajima et al., 2007; Lee et al., 2016), surface and functionalize modification and included the compilation biomolecule features interaction (Ghaemi et al., 2013), in fact there a still remain an ample challenges in promoting and complying the approaches. Ideally the combination should provide chemical stability, physical matching properties, adherent support, and mechanical strength of surface topography and surface modification capacity (Sabine et al., 2008).

The fascinating properties of GO mostly correspondence to its various functional group applicability for instance epoxide, carboxyl and hydroxyl group have prone to amphiphilicity interaction and excellent in transmittance of thin film thickness evenly in bio-composite. Interestingly, that sense, GO ultimately can be functionalized to enhance bioactivity of the substrate or commonly to be used as a surface coating on biomaterials. This is a great promising of surface chemistry modification which serves as a high anticipative tool in regulation of biological responses meantime sustain the stem cell behavior including cell adhesion, viability, proliferation and cell spreading determinate biocompatibility of the certain substrate (Kang et al., 2017: Baker, 2011: Ning et al., 2016). In that reason, we elicited the GO functionalization accordingly to the most common application of stem cell- biomaterial interface illustrated as cell biological application, bio-device, biosensor, bio-environment and so forth.

5.1 Cell biological application

In vitro culture

In general, many of this application frequently based on the unique surface activity between GO-cell cultures which is reflected in physicochemical properties to some extent is amphiphilicity enumerated as hydrophilicity, hydrophobicity, and covalent or non-covalent interaction and to some extent via natural biomaterial mimicry extracellular matrix (ECM). The biological system surface functionalization of GO is therefore pivotal to be construed the solubility and biocompatibility of three ROFG purposely for in vitro cell culture (Makharza et al., 2013: Lin et al., 2011). Some study has prompted to modification on hydrophobicity limitation. There are several methods have delved to alter the interaction of hydrophobicity may include the physically protein adsorption or in short by natural ECM biomaterials interface and covalently modification (Ning et al., 2016: Wu et al., 2006: Vu et al., 2014). To address the modification via physical protein adsorption, hydrophobic surface need to be altered in order to reduce nonspecifically adsorbs serum protein (albumin) from cell which hindered the affinity of cell adhesion. Certainly, Fibronectin (FN), laminin and collagen are the conventional physical protein modification. Basically, chemical modification enables to stimulate the robust interaction between protein and substrate surface (Ning et al., 2016: Kuddannaya et al., 2013). The specific cross-linker possibly to use such as (3-aminopropyl) triehoxy silane (APTES) and cross-linker glutaraldehyde (GA) to modified surface enable cell adhere easily (Lin et al., 2011) especially for cell adhesion and viability of MSCs providing the effective surface modification which reduce the hydrophobicity.

Li et al in 2015 have addressed the huge expression of excellent candidate in incorporating with other materials has essentially fabricated by the functionalized graphene with oxygen-containing chemical group created highly potential as a platform that serve as biocompatible, transferable and implantable device for innovative stem cell culture, additionally there are large surface area for the immobilization culture system where as the substrate enable to modified especially in the application of biomolecules, fluorescent, and drug carrier (Lee et al., 2011). By Kim et al., 2013-explained ITO, functionalized with micrograph via spectro-electrochemical tool C-G.

Atomically, epoxide (-C=C-), hydroxyl group (C=O-OH), and carboxyl group (C (O) OH) (Rahul et al., 2016; Rosa et al., 2016; Lee et al., 2011) have notably ascertained a greater interactions with protein membrane adsorption through covalent, electrostatic, and hydrogen bonding whereas pivotal in accelerate the stem cell behavior especially in adhesion differentiation in osteogenicity activity.

Deep in studied by Mokkapati et al. (2016) was presented that human Adipose Stem Cells (hASCs) positively growth and well proliferate on a new class of membrane integrated with GO and Boron (NaB). The study of integration GO-NaB towards the method of alternatively consume of ethanol (EtOH) for dispersion GO flakes on top of a glass substrate uniformly revealed highly in biocompatibility in relation to squeezed the cost for cultivation of cell and tissue study.

Owing to the fact that dispersion, Liu et al. (2013) in the review also belief the important of functionalization oxygen-containing hydrophilic groups assist high dispersion in water solvent. Correspondence study with Garcia et al. (2016) stated that the successful uniform evidence of 100% monolayer GO flakes revealed by analysis of both is a powerful surface analytical technique by atomic force microscopy (AFM) and scanning electron microscopy (SEM) in outstanding of oxidation and exfoliation GO.

Dramatically revealed from Erickson and the team, have discovered that the specific atomic features have contributed three major regions illustrated as hole, graphitic, and high contrast of oxygen functionalization density which recapitulating the various functionalization applicable (Erickson et al., 2010: Chen et al., 2012). In addition , the actual figure of structurally GO regardless beneficial to stem cell was elucidated that excellent physiochemical properties that can be seen as ultra-large surface areas, abundant hydrophilic groups and outstanding water solubility give rise in amazing effect of manipulation in cell adhesion, cell-cell interaction, and cell spreading which exhibit from the exceptional GO characteristics specifically in mechanical stiffness, nanotopography and large absorption energy capacity (Park et al. 2015).

Functionalization group of GO alternatively discovered by Rahul et al. (2016) illustrated the nucleophilic addition-elimination reaction whereas to insist the uniform dispersion of nanoparticles in resulting of increasing the adhesive factor for cultivation and developed implantation field. To address the amazing result from this method, high density polyethylene hybrid to polyethylene grafted graphene oxide (HDPE/ PE-g-GO) indicated the highest adsorption of protein on the substrate surface reinforcing obviously with GO substratum compared to unmodified HDPE.

Alternatively, overall scientists are straggling an effort to envisioned the multiple novel approach in regulating the acceleration of stem cell behavior which as according to La and the team also followed by Ji and the groups have been first successful in demonstrated the first time of combining layer-by-layer (LBL) technology with the biomimetic mineralization method (La et al., 2014; Ji et al., 2016).

Behind another issue especially in biocompatibility herein stretched in reviewed by Pinto et al. (2013) epitomize the amazing property of GO as excellent in biocompatibility by highlighted stripes issues for instance are in vitro studies of biocompatibility in mammalian cell and bacterial cells as well as Hemocompatibility in remarkable analysis of GO and composites GO materials in order to distinguish the effect of GO employed in biomedical field of implantation and intravenous application.

Moreover, putting in an expedient work, the promising composites studied by Rahul et al. 2016 successful to develop the combination of nano-composites reinforcement by GO with HDPE/PE-g-GO indicate the highly protein adsorption which established well mechanism interaction of electrostatic and hydrophobic interaction with the substrate upon increasing the interfacial adhesion and proliferation cell culture between matrix and filled. The modification technique by reinforced with GO, ideally enhance the growth numbers of hMSCs in analysis of cytocompatibility assessment using osteoblast and MSCs of cell attachment result the high number of mitochondria in the cells. The great quality of cell morphology also disparity between the control of unmodified with GO by the amazing discovery indicates that the great effort and high commitments in bio-composites candidate giving wider chances in regenerative biomedical loaded to orthopedic application evenly projected.

Myoblast differentiation

Study by Chaudhuri et al., 2015 expelled the unique promising of properties GO physically rougher also has enhanced not only osteoblast hence demonstrated that GO composite Poly-caprolactone (GO-PCL) significantly elevated the proliferation and differentiate in to human skeletal muscle cells (hSkMCs).

In the same perspective, Ku and Park, 2012 has demonstrated that GO on amine modified glass substrate was cleaned by Piranha solution and immersed then modified in 3-APTES in anhydrous toluence solution has shown a great capability to proliferate mouse myoblast C2C12. GO-modified glass substrate has delineated succinctly to express specific genes instantly MyoD, myogenin, Troponin T and myosin heavy chain (MHC). The study has demonstrated the ability of GO-modified glass substrate enable to stimulate myogenic differentiation giving the future lightens in tissue engineering application.

Neuronal differentiation

For the purpose of surface modification of GO for neuronal stem cell, Yang et al. (2016) was grounded explain the other way of functionalization technique on substrate before coating GO which as self-assembled monolayer of positively charged 3-aminopropyltriethoxysilane (APTES) employed on the surface by immersed into APTES solution dissolve in distilled water (DI). Contrary, the elaboration relied on consumed of silicone (SiO2) as a substrate for GO fabrication in a reason of typically template applied for stripes of cell cultures due to the power of biocompatibility. Alternating of outstanding idea on replenish the modification by surface hierarchical topographies cue for instance GO patterned substrate significantly exhibited an expression result in neuronal differentiation using neuronal markers (TUJ1 and MAP2) and promoting the focal adhesion development in human neural stem cells (hNSCs). Customarily, due to the electrical interaction upon the group of carboxyl whereas negatively charged with self-assembled monolayer of positively charged on the substrate passionately innovation the line-patterned samples. In the line to confirm the electrophysiological functionalities, Yang and the group were being performed a whole­cell patch clamp analysis. The neuronal cells derived from hNSCs adhered on GPS revealed the voltage-dependent ionic currents and action potentials which are the electrophysiological characteristics treated using sodium channel antagonist tetrodotoxin (TTX) about 5-10min purposely to examine whether the currents and spikes were Na+ channel specific Yang et al., 2016.


In contrary, discovered by Lee et al. (2015) ideally report that reduced GO (rGO) as applied in dental and orthopedic field mediated spontaneously stimulate osteogenic differentiation of MSCs which as composite with hydroxiapatite (HAp). The result revealed that the composites obviously stimulate the multipotency of stem cell culture and showed the superior bioactivity and biocompatibility with the average of particle sizes of HAp and rGO were 1270 ± 476 nm and 438 ± 180 nm, respectively. The study commence the that rGO-HaP has high potential in dental and orthopedic field.

Owing the unique properties of GO, Akhavan et al., 2012 have rendered the study of GO alone interface to dental pulp stem cell. There would be the first study to projected the specific concentration of 1.5mg/ml GO solution has added to the coverslip which enable to allowed the attachment, proliferation and presented the positive expression for all genes tested such as PAX-9, COL 1 and DMP-1. Furthermore, this pioneer study on dental pulp stem cell reckoned that the rougher surface of GO has attracted the stem cell attachment rather than normal coverslip.

The report of studied by Rosa and colleague has expressed that GO alone obtained by chemical exfoliation of graphite using the modified Hummer’s method enable to elevate the growth of dental pulp stem cell (DPSC). The rougher GO surface capable DPSC to express the gene expression of Paired box-9 (PAX-9), Collagen type I (COL 1), Dentin matrix acidic phosphoprotein 1 (DMP-1) and Homo sapiens dentin sialophosphoprotein (DSPP) (Rosa et al., 2016).


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Modifications of Graphene Oxide-based composites biomaterial promote human Wharton’s Jelly derived Mesenchymal Stem Cells. Functionalization and characterization
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Y. Umul Hanim (Author), 2017, Modifications of Graphene Oxide-based composites biomaterial promote human Wharton’s Jelly derived Mesenchymal Stem Cells. Functionalization and characterization, Munich, GRIN Verlag,


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