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The main takeaway from the article: Brady plans every detail of his life so he can play football as long as possible, and he'll do anything he can to get an edge. He diets all year round, takes scheduled naps in the offseason, never misses a workout, eats what his teammates call "birdseed," and does cognitive exercises to keep his brain sharp. Brady struggles to unwind after games and practices. He's still processing, thinking about what's next.

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Bettingen wertheim procedure

Individual floorboards of lower strength may wear out faster and thus lead to a premature exchange of the complete floor. So better not use these boards at all. Scientific tests have shown a direct connection between the density of wood and its strength. The denser the wood is, the stronger and therefore more resistant is the floor.

Thus quality has become measurable, and the customers get lumber of the minimum density they require. An advantage, particularly for large stage floors which are worn out during performances and stage rehearsals. By using this new procedure the life-time of the floor is significantly longer due to its higher strength.

The measuring process has been developed primarily for heavily used floors and is currently offered and integrated into the manufacturing process of only these floors. Especially planners appreciate this important information and security these figures provide. In addition, hMSCs represent suitable non-malignant cells for toxicological investigations of NPs under long-term cultivation conditions, since hMSCs can be expanded over several passages without transformation or immortalization [ 16 , 17 ].

Information about the toxicity of the NPs, as well as functional impairment of hMSCs, is still incomplete and partially contradictory [ 1 , 14 , 17 ]. For instance, it is unclear whether superparamagnetic iron oxide nanoparticles inhibit the chondrogenic differentiation of hMSCs [ 18 ] or not [ 19 ]. Our group previously demonstrated the intracellular accumulation of ZnO-NPs in the cytoplasm of hASCs after a cultivation period of three weeks.

The ASCs showed a significant impairment of cell migration after ZnO-NPs exposure, but no alteration of their multi-differentiation potential in histologic analyses [ 12 ]. The wide range of possible applications of NPs in the stem cell field requires detailed information about the interactions between NPs and human stem cells.

Thus, the purpose of the present study was to evaluate the impact of subtoxic concentrations of ZnO-NPs on cytokine expression and multi-differentiation potential of ASCs, four weeks after exposure. According to transmission electron microscopy TEM , nanoparticles were spherical in shape with a mean diameter of 45—55 nm.

Regarding measurements of the dynamic light scattering, the average diameter of particle aggregates was Immediately after exposure, no ZnO-NPs were detectable within the cells. Particle aggregates were found attached to the cell membrane, however, no inclusion of particles into the cells was observed. Clearly, the intracellular presence of ZnO-NPs within the exposed ASCs could be determined at all other time points one, two, three, and four weeks after exposure.

After one week, TEM patterns still showed some intracellular vesicles in the cytoplasm containing small zinc oxide particles in the exposed ASCs Figure 1. After two weeks and at later time points, no more particle-containing vesicles were found. Instead, free particle aggregates were seen in the cytoplasm or rarely in organelles, but preferably lysosomes Figure 1 B.

No inclusion into the nucleus was assessed. The black arrow indicates a vesicle containing conglomerates of ZnO-NPs. The white arrow indicates single particles in the cytoplasm scale bar represents nm. The scale bar represents nm.

The analyses revealed no impairment of viability compared to the unexposed controls immediately after exposure and seven days, 14 days, 21 days, as well as 28 days after the procedure Figure 2. The analyses revealed no impairment of viability compared to the unexposed controls immediately after exposure and 7, 14, 21 as well as 28 days after the procedure.

The mean extinction values were averaged from 8 wells per patient and normalized to the respective values of the unexposed ASCs of the same patient. Comparing the ZnO-NPs-exposed and unexposed ASCs, there were no remarkable differences apparent in the histological patterns of the differentiation assays.

Typical intracellular lipid vacuoles were detected in both groups after adipogenic differentiation using the Oil Red O stain Figure 3 A. The deposition of extracellular calcium after the osteogenic differentiation of the ZnO-NPs-exposed and unexposed cells was confirmed using the Alizarin Red and von Kossa stains Figure 3 A , whereas the negative controls revealed no extracellular matrix deposition not shown.

A Histologic analyses of adipogenic and osteogenic differentiation in ZnO-NPs exposed and unexposed ASCs: Oil Red O-stained intracellular lipid vacuoles could be detected in both exposed and unexposed cells after three weeks of adipogenic differentiation. Deposition of extracellular calcium deposits was detected in exposed and unexposed cells using Alizarin Red red nodules and von Kossa stain black nodules in both groups after osteogenic differentiation.

Box-whisker plots show the median, 1st quartile, and 3rd quartile, as well as the minimal and maximal values of CT values normalized to GAPDH. In addition, the differentiation potential was confirmed quantitatively by the measurement of gene expression levels of fatty acid binding protein 4 FABP4 , leptin, and lipoproteinlipase LPL , which are specific marker genes for adipogenic differentiation and alkaline phosphatase ALP , Runt-related transcription factor 2 RUNX-2 and osteocalcin bone gamma-carboxylglutamate protein, BGLAP , which indicate osteogenic differentiation.

ZnO-NPs are widely used as an additive to paints and cements, as well as in the pharmaceutical and cosmetic industries. ZnO-NPs are an important ingredient of sunscreens. Consumers, as well as employees in the chemical industry, come into contact with ZnO via skin and airway exposure, respectively [ 1 ]. While the stratum corneum of intact skin may sufficiently protect the organism against NPs, injuries or skin lesions due to chronic skin disease or sunburn may allow the NPs to enter deeper layers of the epidermis with their proliferating cells [ 1 , 20 , 21 ].

Recently, after topical application of ZnO-NPs in vivo, an accumulation within the hair follicles with consecutive apoptosis of the hair follicle stem cells HFSCs was described. Additional in vitro studies revealed DNA damage, impairment of the differentiation potential of the HFSCs and altered expression of marker genes for apoptosis, differentiation and cell communication [ 22 ].

Besides their common use in consumer products, various applications of NPs in stem cell research and medicine are discussed [ 14 ]. Magnetic nanoparticles can be used for tracking and guiding stem cells to their desired site or as a vector for targeted delivery of biotherapeutics [ 14 , 23 ]. ZnO-NPs should be evaluated regarding their potential to sensitize cells to ionizing radiation. In this context, stem cells could be an interesting carrier for particles in order to stay within the tumor formation due to a high migration and adhesion of stem cells towards tumor tissue [ 24 ].

However, the impact of NPs exposure and the intracellular accumulation of NPs long-term after exposure [ 17 , 22 ] on functional capabilities of stem cells remain unclear and partially controversial [ 12 , 18 , 19 , 22 ].

Thus, it was the aim of the present study to provide additional information about the interaction between ZnO-NPs and human adipose-derived stromal cells hASCs with the focus on functional aspects. Our evaluation was not only based on qualitative visual observations using the histological patterns, but also on the quantitative analysis of specific marker gene expression for adipogenic and osteogenic differentiation using real-time PCR.

All experiments were performed using subtoxic concentrations, as shown by preserved viability of the ASCs after 24 h of exposure and after seven, 14, 21 and 28 days, as confirmed by the MTT assay. After differentiation under specific medium conditions for three weeks, no differences between ZnO-NPs-exposed ASCs and non-exposed cells were apparent in the histological patterns.

Gene expression of specific adipogenic and osteogenic marker genes did not significantly differ in both groups. Others described alterations of differentiation marker gene expression in HFSCs after exposure to ZnO-NPs, whereas a distinct concentration and application of ZnO-NPs, as well as a different cell type, were used for the experiments [ 22 ].

Nevertheless, recovery time after the exposure procedure may facilitate certain repair processes within the exposed cells. However, histologic analyses previously performed did not reveal impaired multi-differentiation potential of the hASCs, which underwent the differentiation procedures immediately after 24 h of ZnO-NPs exposure. However, an impairment of migration capability was observed for exposed hASCs in this study [ 12 ]. Thus, a decreased compromising effect of ZnO-NPs during the differentiation procedure can be assumed.

This is contrary to results of our previous study with silver nanoparticles Ag-NPs [ 25 ] and the observation of others [ 26 ], who demonstrated an increased secretion of IL-8 as an indicator for stem cell activation in vascular endothelial cells after short-term ZnO-NPs exposure.

Besides the mediation of stem cell activation, IL-6 and IL-8 are also well-known chemotactic cytokines for the attraction of neutrophils, eosinophils, and T lymphocytes [ 27 ]. Elevated levels of these cytokines may induce inflammation in several tissue models after NPs exposure. Such inflammatory potential was already demonstrated for ZnO-NPs [ 28 ]. In addition, ZnO-NPs are suspected to induce a chronic pro-inflammatory milieu in the human upper aerodigestive tract [ 29 ].

Although NPs were intracellularly trapped in the ASCs over the whole period of observation four weeks , the cytokine gene expression was not affected. The main difference to other studies, is that we applied a safe and surely non-toxic amount of NPs instead of borderline concentrations, as done in the other studies.

Thus, this finding clearly underlines the fact, that the functionality of the cells is likely to be unaffected by ZnO-NPs at low concentrations. In addition, a long-term disposition of NPs in the cells does not influence their functionality, supposing that the starting concentration was safely in the non-toxic range.

It is well-known, that the shape and size of the nanoparticles critically influence their toxicity [ 1 , 30 , 31 , 32 , 33 ]. In the current study, we chose one representative nanoparticle size, since our experiments focused on the effect of time on toxicity and functionality.

However, physical properties like shape and size must be assessed as well, since data on nanotoxicology of different sized ZnO-NPs in ASCs are lacking. Theories of ongoing intracellular zinc ion release from NP-aggregates is not supported by our findings. This might be an important information for single-use applications of ZnO-NPs on nanomedical approaches. Cells with a short life span will probably release particles during apoptosis, so they will be removed by dendritic cells or macrophages.

However, long-living cells, like stem cells, are likely to contain nanoparticles over a long time. In the opinion of the authors, the application of ZnO-NPs under subtoxic concentrations will be safe, even if NPs remain in the cells over a long period. However, repetitive exposure to such low concentrations will surely lead to further NPs accumulation in the cells and studies using repeated NPs exposures are especially interesting in long-living cell types.

The dispersion was stabilized and neutralized according to Bihari et al. Dynamic light scattering Malvern Instruments Ltd. To study the ultrastructure of intracellular particle distribution, as well as to quantify particle-containing cells during expansion, TEM was performed as described previously [ 29 ]. Pellets containing ASCs of three patients each were fixed for 30 min in a solution of 0. Afterwards, staining was performed overnight with 0. The specimens were dehydrated, embedded in epoxy resin Epon , cut into ultrathin sections of 60 nm thickness and examined on a Zeiss transmission electron microscope EM Carl Zeiss AG, Oberkochen, Germany at the Division of Electron Microscopy group of Prof.

The photographic negatives were digitalized by scanning. The ASCs were isolated from the adipose tissue of 6 healthy donors with their informed consent. The isolation procedure was previously described [ 16 ]. After centrifugation and discarding the supernatant, including the adipose tissue, erythrocyte lysis buffer mM ammonium chloride NH 4 Cl , 10 mM potassium bicarbonate KHCO3 , 0.

The isolated and plated cells were defined as passage 0. The medium was replaced every third day during expansion. The cells were detached with 0. They were defined as passage 1. Immediately afterwards, the viability of the hASCs was evaluated using the MTT [3- 4,5-dimethylthiazolyl -2,5-diphenyl tetrazolium bromide] colorimetric staining method [ 36 , 37 ].

The remaining cells were resuspended in expansion medium and cultured for 4 weeks without any additional exposure to ZnO-NPs during this time course. Untreated hASCs served as negative controls. Eight wells were seeded for each of the six patients at each time point. Viability of exposed cells and positive control was indicated as a percentage of the viability of the unexposed controls.

In order to determine a possible impairment of the multilineage differentiation potential of the hASCs due to ZnO-NPs exposure, adipogenic and osteogenic differentiation procedures were performed, as previously described [ 30 ]. Human ASCs of the six patients were used for the multilineage differentiation studies four weeks after the exposure procedure hASCs of passage 6.

Human ASCs were maintained in the defined media for three weeks and the medium was replaced every other day. The adipogenic and osteogenic differentiation was confirmed qualitatively using histology and quantitatively using real time-polymerase chain reaction PCR analyses. To show intracellular lipid vacuoles after adipogenic differentiation, the Oil Red O stain was used.

Von Kossa staining revealed extracellular mineral deposition after osteogenic differentiation by the presence of black nodules. In addition, extracellular calcium deposits were stained red using the Alizarin Red solution. Adipogenic and osteogenic differentiation was confirmed by the measurement of lineage-specific gene expression. For comparison of gene expression of the ZnO-NPs-exposed and unexposed hASCs after multilineage differentiation procedures, the unpaired t -test was used when Gaussian distribution could be confirmed Figure 3.

Otherwise, the Mann—Whitney U test was applied Figure 3. Significance is indicated in the figures by asterisks. The results were mostly charted using Boxplots. The box shows the median, the 1st quartile, and the 3rd quartile, and the whiskers represent the minimal and maximal values.

The columns show the mean and standard error of the mean SEM. Every author has contributed substantially to the work reported. Read article at publisher's site DOI : Nanomaterials Basel , 10 4 , 13 Apr Materials Basel , 12 24 , 05 Dec To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.

Cell J , 17 3 , 07 Oct J Biomed Nanotechnol , 9 1 , 01 Jan Cited by: 22 articles PMID: Crit Rev Toxicol , 46 4 , 25 Feb Cited by: 29 articles PMID: J Anim Sci Biotechnol , , 09 Jul Coronavirus: Find the latest articles and preprints. Europe PMC requires Javascript to function effectively.

Recent Activity. Recent history Saved searches. Abstract Free full text 1. Introduction 2. Results 3. Discussion 4. Radeloff K 1 ,. Radeloff A 1 ,. Tirado MR 2 ,. Scherzad A 2 ,. Hagen R 2 ,. Kleinsasser kepleruniklinikum. Kleinsasser NH 3 ,. Hackenberg S 2. Affiliations 2 authors 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook.

Free full text.

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The lumber, of course, must be adapted to additional strains like heavy rolling loads or the use of fork lifters. This can be achieved by an even more precise sorting of the lumber. So far the choice of lumber for a stage floor has been a very subjective process. However, this method was not objective. A gradual structural change may remain unnoticed.

Individual floorboards of lower strength may wear out faster and thus lead to a premature exchange of the complete floor. So better not use these boards at all. Scientific tests have shown a direct connection between the density of wood and its strength.

The denser the wood is, the stronger and therefore more resistant is the floor. Nevertheless, recovery time after the exposure procedure may facilitate certain repair processes within the exposed cells. However, histologic analyses previously performed did not reveal impaired multi-differentiation potential of the hASCs, which underwent the differentiation procedures immediately after 24 h of ZnO-NPs exposure.

However, an impairment of migration capability was observed for exposed hASCs in this study [ 12 ]. Thus, a decreased compromising effect of ZnO-NPs during the differentiation procedure can be assumed. This is contrary to results of our previous study with silver nanoparticles Ag-NPs [ 25 ] and the observation of others [ 26 ], who demonstrated an increased secretion of IL-8 as an indicator for stem cell activation in vascular endothelial cells after short-term ZnO-NPs exposure.

Besides the mediation of stem cell activation, IL-6 and IL-8 are also well-known chemotactic cytokines for the attraction of neutrophils, eosinophils, and T lymphocytes [ 27 ]. Elevated levels of these cytokines may induce inflammation in several tissue models after NPs exposure. Such inflammatory potential was already demonstrated for ZnO-NPs [ 28 ].

In addition, ZnO-NPs are suspected to induce a chronic pro-inflammatory milieu in the human upper aerodigestive tract [ 29 ]. Although NPs were intracellularly trapped in the ASCs over the whole period of observation four weeks , the cytokine gene expression was not affected.

The main difference to other studies, is that we applied a safe and surely non-toxic amount of NPs instead of borderline concentrations, as done in the other studies. Thus, this finding clearly underlines the fact, that the functionality of the cells is likely to be unaffected by ZnO-NPs at low concentrations.

In addition, a long-term disposition of NPs in the cells does not influence their functionality, supposing that the starting concentration was safely in the non-toxic range. It is well-known, that the shape and size of the nanoparticles critically influence their toxicity [ 1 , 30 , 31 , 32 , 33 ]. In the current study, we chose one representative nanoparticle size, since our experiments focused on the effect of time on toxicity and functionality. However, physical properties like shape and size must be assessed as well, since data on nanotoxicology of different sized ZnO-NPs in ASCs are lacking.

Theories of ongoing intracellular zinc ion release from NP-aggregates is not supported by our findings. This might be an important information for single-use applications of ZnO-NPs on nanomedical approaches. Cells with a short life span will probably release particles during apoptosis, so they will be removed by dendritic cells or macrophages.

However, long-living cells, like stem cells, are likely to contain nanoparticles over a long time. In the opinion of the authors, the application of ZnO-NPs under subtoxic concentrations will be safe, even if NPs remain in the cells over a long period.

However, repetitive exposure to such low concentrations will surely lead to further NPs accumulation in the cells and studies using repeated NPs exposures are especially interesting in long-living cell types. The dispersion was stabilized and neutralized according to Bihari et al.

Dynamic light scattering Malvern Instruments Ltd. To study the ultrastructure of intracellular particle distribution, as well as to quantify particle-containing cells during expansion, TEM was performed as described previously [ 29 ]. Pellets containing ASCs of three patients each were fixed for 30 min in a solution of 0. Afterwards, staining was performed overnight with 0. The specimens were dehydrated, embedded in epoxy resin Epon , cut into ultrathin sections of 60 nm thickness and examined on a Zeiss transmission electron microscope EM Carl Zeiss AG, Oberkochen, Germany at the Division of Electron Microscopy group of Prof.

The photographic negatives were digitalized by scanning. The ASCs were isolated from the adipose tissue of 6 healthy donors with their informed consent. The isolation procedure was previously described [ 16 ]. After centrifugation and discarding the supernatant, including the adipose tissue, erythrocyte lysis buffer mM ammonium chloride NH 4 Cl , 10 mM potassium bicarbonate KHCO3 , 0.

The isolated and plated cells were defined as passage 0. The medium was replaced every third day during expansion. The cells were detached with 0. They were defined as passage 1. Immediately afterwards, the viability of the hASCs was evaluated using the MTT [3- 4,5-dimethylthiazolyl -2,5-diphenyl tetrazolium bromide] colorimetric staining method [ 36 , 37 ].

The remaining cells were resuspended in expansion medium and cultured for 4 weeks without any additional exposure to ZnO-NPs during this time course. Untreated hASCs served as negative controls. Eight wells were seeded for each of the six patients at each time point. Viability of exposed cells and positive control was indicated as a percentage of the viability of the unexposed controls.

In order to determine a possible impairment of the multilineage differentiation potential of the hASCs due to ZnO-NPs exposure, adipogenic and osteogenic differentiation procedures were performed, as previously described [ 30 ]. Human ASCs of the six patients were used for the multilineage differentiation studies four weeks after the exposure procedure hASCs of passage 6. Human ASCs were maintained in the defined media for three weeks and the medium was replaced every other day.

The adipogenic and osteogenic differentiation was confirmed qualitatively using histology and quantitatively using real time-polymerase chain reaction PCR analyses. To show intracellular lipid vacuoles after adipogenic differentiation, the Oil Red O stain was used. Von Kossa staining revealed extracellular mineral deposition after osteogenic differentiation by the presence of black nodules. In addition, extracellular calcium deposits were stained red using the Alizarin Red solution. Adipogenic and osteogenic differentiation was confirmed by the measurement of lineage-specific gene expression.

For comparison of gene expression of the ZnO-NPs-exposed and unexposed hASCs after multilineage differentiation procedures, the unpaired t -test was used when Gaussian distribution could be confirmed Figure 3. Otherwise, the Mann—Whitney U test was applied Figure 3. Significance is indicated in the figures by asterisks.

The results were mostly charted using Boxplots. The box shows the median, the 1st quartile, and the 3rd quartile, and the whiskers represent the minimal and maximal values. The columns show the mean and standard error of the mean SEM. Every author has contributed substantially to the work reported.

Read article at publisher's site DOI : Nanomaterials Basel , 10 4 , 13 Apr Materials Basel , 12 24 , 05 Dec To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.

Cell J , 17 3 , 07 Oct J Biomed Nanotechnol , 9 1 , 01 Jan Cited by: 22 articles PMID: Crit Rev Toxicol , 46 4 , 25 Feb Cited by: 29 articles PMID: J Anim Sci Biotechnol , , 09 Jul Coronavirus: Find the latest articles and preprints. Europe PMC requires Javascript to function effectively. Recent Activity. Recent history Saved searches. Abstract Free full text 1. Introduction 2. Results 3. Discussion 4. Radeloff K 1 ,. Radeloff A 1 ,.

Tirado MR 2 ,. Scherzad A 2 ,. Hagen R 2 ,. Kleinsasser kepleruniklinikum. Kleinsasser NH 3 ,. Hackenberg S 2. Affiliations 2 authors 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Free full text. Materials Basel. Published online Jun 5. PMID: Kleinsasser , 3 and Stephan Hackenberg 2. Find articles by Mario Ramos Tirado. Find articles by Agmal Scherzad. Find articles by Rudolf Hagen. Norbert H.

Find articles by Stephan Hackenberg. Author information Article notes Copyright and License information Disclaimer. Received Apr 30; Accepted Jun 4. This article has been cited by other articles in PMC. Go to:. Keywords: zinc oxide, nanoparticles, toxicity, differentiation potential, human adipose-derived stromal cells, stem cells.

Characterization of Nanoparticles According to transmission electron microscopy TEM , nanoparticles were spherical in shape with a mean diameter of 45—55 nm. Open in a separate window. Figure 1. Figure 2. Multilineage Differentiation Potential 2. Histology Comparing the ZnO-NPs-exposed and unexposed ASCs, there were no remarkable differences apparent in the histological patterns of the differentiation assays. Figure 3. Real Time-PCR Analyses In addition, the differentiation potential was confirmed quantitatively by the measurement of gene expression levels of fatty acid binding protein 4 FABP4 , leptin, and lipoproteinlipase LPL , which are specific marker genes for adipogenic differentiation and alkaline phosphatase ALP , Runt-related transcription factor 2 RUNX-2 and osteocalcin bone gamma-carboxylglutamate protein, BGLAP , which indicate osteogenic differentiation.

Figure 4. Multilineage Differentiation Potential In order to determine a possible impairment of the multilineage differentiation potential of the hASCs due to ZnO-NPs exposure, adipogenic and osteogenic differentiation procedures were performed, as previously described [ 30 ]. PCR Analyses Adipogenic and osteogenic differentiation was confirmed by the measurement of lineage-specific gene expression. Scherzad A. Rasmussen J. Zinc oxide nanoparticles for selective destruction of tumor cells and potential for drug delivery applications.

Expert Opin. Drug Deliv. Ramos A. Biomedical Applications of Nanotechnology. Hackenberg S. Zinc oxide nanoparticles induce photocatalytic cell death in human head and neck squamous cell carcinoma cell lines in vitro. Oedayrajsingh-Varma M. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.

Zuk P. Human adipose tissue is a source of multipotent stem cells. Froelich K. Adipose-derived stromal cells ASC —Basics and therapeutic approaches in otorhinolaryngology. Zielins E. Therapeutic applications of human adipose-derived stromal cells for soft tissue reconstruction. Adipose-derived stem cells: Sources, potency, and implications for regenerative therapies. Matsumoto D. Cell-assisted lipotransfer: Supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection.

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The main difference to other studies, is that we applied a safe and surely non-toxic amount of NPs instead of borderline concentrations, as done in the other studies. Thus, this finding clearly underlines the fact, that the functionality of the cells is likely to be unaffected by ZnO-NPs at low concentrations. In addition, a long-term disposition of NPs in the cells does not influence their functionality, supposing that the starting concentration was safely in the non-toxic range.

It is well-known, that the shape and size of the nanoparticles critically influence their toxicity [ 1 , 30 , 31 , 32 , 33 ]. In the current study, we chose one representative nanoparticle size, since our experiments focused on the effect of time on toxicity and functionality. However, physical properties like shape and size must be assessed as well, since data on nanotoxicology of different sized ZnO-NPs in ASCs are lacking.

Theories of ongoing intracellular zinc ion release from NP-aggregates is not supported by our findings. This might be an important information for single-use applications of ZnO-NPs on nanomedical approaches. Cells with a short life span will probably release particles during apoptosis, so they will be removed by dendritic cells or macrophages. However, long-living cells, like stem cells, are likely to contain nanoparticles over a long time. In the opinion of the authors, the application of ZnO-NPs under subtoxic concentrations will be safe, even if NPs remain in the cells over a long period.

However, repetitive exposure to such low concentrations will surely lead to further NPs accumulation in the cells and studies using repeated NPs exposures are especially interesting in long-living cell types. The dispersion was stabilized and neutralized according to Bihari et al. Dynamic light scattering Malvern Instruments Ltd. To study the ultrastructure of intracellular particle distribution, as well as to quantify particle-containing cells during expansion, TEM was performed as described previously [ 29 ].

Pellets containing ASCs of three patients each were fixed for 30 min in a solution of 0. Afterwards, staining was performed overnight with 0. The specimens were dehydrated, embedded in epoxy resin Epon , cut into ultrathin sections of 60 nm thickness and examined on a Zeiss transmission electron microscope EM Carl Zeiss AG, Oberkochen, Germany at the Division of Electron Microscopy group of Prof. The photographic negatives were digitalized by scanning.

The ASCs were isolated from the adipose tissue of 6 healthy donors with their informed consent. The isolation procedure was previously described [ 16 ]. After centrifugation and discarding the supernatant, including the adipose tissue, erythrocyte lysis buffer mM ammonium chloride NH 4 Cl , 10 mM potassium bicarbonate KHCO3 , 0.

The isolated and plated cells were defined as passage 0. The medium was replaced every third day during expansion. The cells were detached with 0. They were defined as passage 1. Immediately afterwards, the viability of the hASCs was evaluated using the MTT [3- 4,5-dimethylthiazolyl -2,5-diphenyl tetrazolium bromide] colorimetric staining method [ 36 , 37 ]. The remaining cells were resuspended in expansion medium and cultured for 4 weeks without any additional exposure to ZnO-NPs during this time course.

Untreated hASCs served as negative controls. Eight wells were seeded for each of the six patients at each time point. Viability of exposed cells and positive control was indicated as a percentage of the viability of the unexposed controls. In order to determine a possible impairment of the multilineage differentiation potential of the hASCs due to ZnO-NPs exposure, adipogenic and osteogenic differentiation procedures were performed, as previously described [ 30 ].

Human ASCs of the six patients were used for the multilineage differentiation studies four weeks after the exposure procedure hASCs of passage 6. Human ASCs were maintained in the defined media for three weeks and the medium was replaced every other day. The adipogenic and osteogenic differentiation was confirmed qualitatively using histology and quantitatively using real time-polymerase chain reaction PCR analyses. To show intracellular lipid vacuoles after adipogenic differentiation, the Oil Red O stain was used.

Von Kossa staining revealed extracellular mineral deposition after osteogenic differentiation by the presence of black nodules. In addition, extracellular calcium deposits were stained red using the Alizarin Red solution. Adipogenic and osteogenic differentiation was confirmed by the measurement of lineage-specific gene expression. For comparison of gene expression of the ZnO-NPs-exposed and unexposed hASCs after multilineage differentiation procedures, the unpaired t -test was used when Gaussian distribution could be confirmed Figure 3.

Otherwise, the Mann—Whitney U test was applied Figure 3. Significance is indicated in the figures by asterisks. The results were mostly charted using Boxplots. The box shows the median, the 1st quartile, and the 3rd quartile, and the whiskers represent the minimal and maximal values.

The columns show the mean and standard error of the mean SEM. Every author has contributed substantially to the work reported. Read article at publisher's site DOI : Nanomaterials Basel , 10 4 , 13 Apr Materials Basel , 12 24 , 05 Dec To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. Cell J , 17 3 , 07 Oct J Biomed Nanotechnol , 9 1 , 01 Jan Cited by: 22 articles PMID: Crit Rev Toxicol , 46 4 , 25 Feb Cited by: 29 articles PMID: J Anim Sci Biotechnol , , 09 Jul Coronavirus: Find the latest articles and preprints.

Europe PMC requires Javascript to function effectively. Recent Activity. Recent history Saved searches. Abstract Free full text 1. Introduction 2. Results 3. Discussion 4. Radeloff K 1 ,. Radeloff A 1 ,. Tirado MR 2 ,. Scherzad A 2 ,. Hagen R 2 ,. Kleinsasser kepleruniklinikum. Kleinsasser NH 3 ,. Hackenberg S 2. Affiliations 2 authors 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Free full text. Materials Basel. Published online Jun 5. PMID: Kleinsasser , 3 and Stephan Hackenberg 2.

Find articles by Mario Ramos Tirado. Find articles by Agmal Scherzad. Find articles by Rudolf Hagen. Norbert H. Find articles by Stephan Hackenberg. Author information Article notes Copyright and License information Disclaimer. Received Apr 30; Accepted Jun 4. This article has been cited by other articles in PMC. Go to:. Keywords: zinc oxide, nanoparticles, toxicity, differentiation potential, human adipose-derived stromal cells, stem cells.

Characterization of Nanoparticles According to transmission electron microscopy TEM , nanoparticles were spherical in shape with a mean diameter of 45—55 nm. Open in a separate window. Figure 1. Figure 2. Multilineage Differentiation Potential 2. Histology Comparing the ZnO-NPs-exposed and unexposed ASCs, there were no remarkable differences apparent in the histological patterns of the differentiation assays. Figure 3. Real Time-PCR Analyses In addition, the differentiation potential was confirmed quantitatively by the measurement of gene expression levels of fatty acid binding protein 4 FABP4 , leptin, and lipoproteinlipase LPL , which are specific marker genes for adipogenic differentiation and alkaline phosphatase ALP , Runt-related transcription factor 2 RUNX-2 and osteocalcin bone gamma-carboxylglutamate protein, BGLAP , which indicate osteogenic differentiation.

Figure 4. Multilineage Differentiation Potential In order to determine a possible impairment of the multilineage differentiation potential of the hASCs due to ZnO-NPs exposure, adipogenic and osteogenic differentiation procedures were performed, as previously described [ 30 ]. PCR Analyses Adipogenic and osteogenic differentiation was confirmed by the measurement of lineage-specific gene expression.

Scherzad A. Rasmussen J. Zinc oxide nanoparticles for selective destruction of tumor cells and potential for drug delivery applications. Expert Opin. Drug Deliv. Ramos A. Biomedical Applications of Nanotechnology. Hackenberg S. Zinc oxide nanoparticles induce photocatalytic cell death in human head and neck squamous cell carcinoma cell lines in vitro.

Oedayrajsingh-Varma M. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Zuk P. Human adipose tissue is a source of multipotent stem cells. Froelich K. Adipose-derived stromal cells ASC —Basics and therapeutic approaches in otorhinolaryngology. Zielins E. Therapeutic applications of human adipose-derived stromal cells for soft tissue reconstruction.

Adipose-derived stem cells: Sources, potency, and implications for regenerative therapies. Matsumoto D. Cell-assisted lipotransfer: Supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection.

Tissue Eng. Yoshimura K. Cell-assisted lipotransfer for facial lipoatrophy: Efficacy of clinical use of adipose-derived stem cells. Roger M. Mesenchymal stem cells as cellular vehicles for delivery of nanoparticles to brain tumors. El-Sadik A. Kang H. Magnetic bionanoparticle enhances homing of endothelial progenitor cells in mouse hindlimb ischemia. Korean Circ. Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro.

Ickrath P. Drop Bags Taubertal Assemble at the front of Hotel Rappen. Runners carry burning torches for a group run through the old town to the castle garden for the knight's breefing. Run with burning torches. Final Briefing by a Knight on Horseback.

The knight gives the final briefing. Downhill run on a single trail to the start area in the Taubertal-Valley. Start of the Taubertal The start is at the "Eiswiese" on the Taubertalweg. The 50, 71, km and mile runners start together. Checkpoints with Food and Drinks:. Drink stations: Water, apple juice, tea, beer, electrolytes.

Food stations: Fruit, soup, mashed potato, yoghurt, chia pudding, Himalayan-salt, coconut oil. Checkpoint with drinks and food. Taubertal checkpoint map for 50 km, 71 km, km and miles:. Exact locations can be seen on the GPS file. Expected arrival times for km-runners with 7-, and hour finishing times, fasted 50 km runner and slowest mile runner. Rothenburg to Wertheim:. Bad Mergentheim. Time Limits:. The first runners will be expected after hours at Saturday around a. The first runners will be expected after 7 hours at Saturday around a.

The first runners will be expected after 13 hours at Saturday around p. Runners who exceed any of the cut-off times will be disqualified. Running speed:. Finish for 50 km runners in Bad Mergentheim. Showers are available in Bad Mergentheim. Onwards transport to Wertheim by train. A shuttle bus back to Rothenburg is available at p. Pick-up at the train station. Finish 71 km runners in Tauberbischofsheim.

Showers are available in Tauberbischofsheim. Finish km runners in Wertheim. Showers are available in Wertheim in the sports hall. Finisher of the km race. Knighting ceremony for every km finisher. Medal for km Finishers. Bus Transport from Wertheim to Rothenburg. Departs from the train station. Welcome Party for all Runners in the Castle of Wertheim. Those not wishing to partake of the Knight's Dinner, may order off the menu or simply buy a drink. The five course Knight's Dinner is available as classic or vegetarian.

The first course of the Knight's Dinner will be served from - p. Classic Knight's Dinner:. First course: Spelt bread and butter. Second course: Selection of salads. Third course: Green spelt soup in wooden bowls.

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