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Effects of dendritic core-shell glycoarchitectures on primary mesenchymal stem cells and osteoblasts obtained from different human donors.

Lautenschläger S, Striegler C, Dakischew O, Schütz I, Szalay G, Schnettler R, Heiß C, Appelhans D, Lips KS - J Nanobiotechnology (2015)

Bottom Line: As well dose as incubation-time dependent effects and interactions have been researched for concentrations between 1 μg/ml to 1 mg/ml and periods of 24 h up to 28 days.In all experiments PEI-5k-Mal-B exhibits a superior biocompatibility compared to PEI-25k-Mal-B.Additionally, for all experiments, results are strongly influenced by a large donor-to-donor variability of the four different rdMSC samples.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Trauma Surgery, Justus-Liebig-University Giessen, Schubertstr. 81, 35392, Giessen, Germany. Stefan.Lautenschlaeger@med.uni-giessen.de.

ABSTRACT
The biological impact of novel nano-scaled drug delivery vehicles in highly topical therapies of bone diseases have to be investigated in vitro before starting in vivo trials. Highly desired features for these materials are a good cellular uptake, large transport capacity for drugs and a good bio-compatibility. Essentially the latter has to be addressed as first point on the agenda. We present a study on the biological interaction of maltose-modified poly(ethyleneimine) (PEI-Mal) on primary human mesenchymal stem cell, harvested from reaming debris (rdMSC) and osteoblasts obtained from four different male donors. PEI-Mal-nanoparticles with two different molecular weights of the PEI core (5000 g/mol for PEI-5k-Mal-B and 25,000 g/mol for PEI-25k-Mal-B) have been administered to both cell lines. As well dose as incubation-time dependent effects and interactions have been researched for concentrations between 1 μg/ml to 1 mg/ml and periods of 24 h up to 28 days. Studies conducted by different methods of microscopy as light microscopy, fluorescence microscopy, transmission-electron-microscopy and quantitative assays (LDH and DC-protein) indicate as well a good cellular uptake of the nanoparticles as a particle- and concentration-dependent impact on the cellular macro- and micro-structure of the rdMSC samples. In all experiments PEI-5k-Mal-B exhibits a superior biocompatibility compared to PEI-25k-Mal-B. At higher concentrations PEI-25k-Mal-B is toxic and induces a directly observable mitochondrial damage. The alkaline phosphatase assay (ALP), has been conducted to check on the possible influence of nanoparticles on the differentiation capabilities of rdMSC to osteoblasts. In addition the production of mineralized matrix has been shown by von-Kossa stained samples. No influence of the nanoparticles on the ALP per cell has been detected. Additionally, for all experiments, results are strongly influenced by a large donor-to-donor variability of the four different rdMSC samples. To summarize, while featuring a good cellular uptake, PEI-5k-Mal-B induces only minimal adverse effects and features clearly superior biocompatibility compared to the larger PEI-25k-Mal-B.

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a Total cell count of osteogenic differentiated cells determined by DC-protein-measurements. After 28 days a significant reduction in cell count is observed for all concentrations of PEI-25k-Mal-B and for the higher and the intermediate concentration of PEI-5k-Mal-B (***p < 0.001, **p < 0.01). b Normalized ALP content per cell of rdMSC differentiated to osteoblasts and incubated with different concentrations of both nanoparticles. The measured total ALP concentration has been divided by the cell count. As reference for normalization the respective control samples for each endpoint have been used. No influence of nanoparticles on the ALP concentration per cell has been detected
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Fig5: a Total cell count of osteogenic differentiated cells determined by DC-protein-measurements. After 28 days a significant reduction in cell count is observed for all concentrations of PEI-25k-Mal-B and for the higher and the intermediate concentration of PEI-5k-Mal-B (***p < 0.001, **p < 0.01). b Normalized ALP content per cell of rdMSC differentiated to osteoblasts and incubated with different concentrations of both nanoparticles. The measured total ALP concentration has been divided by the cell count. As reference for normalization the respective control samples for each endpoint have been used. No influence of nanoparticles on the ALP concentration per cell has been detected

Mentions: The influence of the nanoparticles on the osteogenic differentiation of rdMSC has been investigated by measuring the ALP content of the cells after 7, 21 and 28 days as endpoints (Fig. 5b). In consideration of the results of the proliferation tests (Fig. 4) decreasing concentrations of dendritic core–shell glycoarchitectures (0.05 mg/ml = 50, 20 and 1 μg/ml) have been selected. As demonstrated in Fig. 5b, the particles, no matter at what concentration, induce no change of the measured normalized ALP concentration while the proliferation of osteogenic differentiated cells is reduced (Fig. 5a). The latter one is comparable to the results obtained from the rdMSC-experiments (Fig. 4). In addition von-Kossa stained samples of osteogenic differentiated rdMSC, with and without nanoparticles, have been prepared. Figure 6 shows large structures of mineralized matrix, deep black in the image, which hint on excessive production of mineralized matrix and thus on a successful osteogenic differentiation for all investigated conditions, weather incubated with or without nanoparticles. The histomorphometric evaluation of the von-Kossa stained samples exhibited no statistically significant deviations from the reference samples for all investigated conditions. However, while not significant, for the larger particle PEI-25k-Mal-B possibly a trend towards higher mineralization can be found. For all conditions the addition of nanoparticles did not lead to reduced osteogenesis.Fig. 5


Effects of dendritic core-shell glycoarchitectures on primary mesenchymal stem cells and osteoblasts obtained from different human donors.

Lautenschläger S, Striegler C, Dakischew O, Schütz I, Szalay G, Schnettler R, Heiß C, Appelhans D, Lips KS - J Nanobiotechnology (2015)

a Total cell count of osteogenic differentiated cells determined by DC-protein-measurements. After 28 days a significant reduction in cell count is observed for all concentrations of PEI-25k-Mal-B and for the higher and the intermediate concentration of PEI-5k-Mal-B (***p < 0.001, **p < 0.01). b Normalized ALP content per cell of rdMSC differentiated to osteoblasts and incubated with different concentrations of both nanoparticles. The measured total ALP concentration has been divided by the cell count. As reference for normalization the respective control samples for each endpoint have been used. No influence of nanoparticles on the ALP concentration per cell has been detected
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4597403&req=5

Fig5: a Total cell count of osteogenic differentiated cells determined by DC-protein-measurements. After 28 days a significant reduction in cell count is observed for all concentrations of PEI-25k-Mal-B and for the higher and the intermediate concentration of PEI-5k-Mal-B (***p < 0.001, **p < 0.01). b Normalized ALP content per cell of rdMSC differentiated to osteoblasts and incubated with different concentrations of both nanoparticles. The measured total ALP concentration has been divided by the cell count. As reference for normalization the respective control samples for each endpoint have been used. No influence of nanoparticles on the ALP concentration per cell has been detected
Mentions: The influence of the nanoparticles on the osteogenic differentiation of rdMSC has been investigated by measuring the ALP content of the cells after 7, 21 and 28 days as endpoints (Fig. 5b). In consideration of the results of the proliferation tests (Fig. 4) decreasing concentrations of dendritic core–shell glycoarchitectures (0.05 mg/ml = 50, 20 and 1 μg/ml) have been selected. As demonstrated in Fig. 5b, the particles, no matter at what concentration, induce no change of the measured normalized ALP concentration while the proliferation of osteogenic differentiated cells is reduced (Fig. 5a). The latter one is comparable to the results obtained from the rdMSC-experiments (Fig. 4). In addition von-Kossa stained samples of osteogenic differentiated rdMSC, with and without nanoparticles, have been prepared. Figure 6 shows large structures of mineralized matrix, deep black in the image, which hint on excessive production of mineralized matrix and thus on a successful osteogenic differentiation for all investigated conditions, weather incubated with or without nanoparticles. The histomorphometric evaluation of the von-Kossa stained samples exhibited no statistically significant deviations from the reference samples for all investigated conditions. However, while not significant, for the larger particle PEI-25k-Mal-B possibly a trend towards higher mineralization can be found. For all conditions the addition of nanoparticles did not lead to reduced osteogenesis.Fig. 5

Bottom Line: As well dose as incubation-time dependent effects and interactions have been researched for concentrations between 1 μg/ml to 1 mg/ml and periods of 24 h up to 28 days.In all experiments PEI-5k-Mal-B exhibits a superior biocompatibility compared to PEI-25k-Mal-B.Additionally, for all experiments, results are strongly influenced by a large donor-to-donor variability of the four different rdMSC samples.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Trauma Surgery, Justus-Liebig-University Giessen, Schubertstr. 81, 35392, Giessen, Germany. Stefan.Lautenschlaeger@med.uni-giessen.de.

ABSTRACT
The biological impact of novel nano-scaled drug delivery vehicles in highly topical therapies of bone diseases have to be investigated in vitro before starting in vivo trials. Highly desired features for these materials are a good cellular uptake, large transport capacity for drugs and a good bio-compatibility. Essentially the latter has to be addressed as first point on the agenda. We present a study on the biological interaction of maltose-modified poly(ethyleneimine) (PEI-Mal) on primary human mesenchymal stem cell, harvested from reaming debris (rdMSC) and osteoblasts obtained from four different male donors. PEI-Mal-nanoparticles with two different molecular weights of the PEI core (5000 g/mol for PEI-5k-Mal-B and 25,000 g/mol for PEI-25k-Mal-B) have been administered to both cell lines. As well dose as incubation-time dependent effects and interactions have been researched for concentrations between 1 μg/ml to 1 mg/ml and periods of 24 h up to 28 days. Studies conducted by different methods of microscopy as light microscopy, fluorescence microscopy, transmission-electron-microscopy and quantitative assays (LDH and DC-protein) indicate as well a good cellular uptake of the nanoparticles as a particle- and concentration-dependent impact on the cellular macro- and micro-structure of the rdMSC samples. In all experiments PEI-5k-Mal-B exhibits a superior biocompatibility compared to PEI-25k-Mal-B. At higher concentrations PEI-25k-Mal-B is toxic and induces a directly observable mitochondrial damage. The alkaline phosphatase assay (ALP), has been conducted to check on the possible influence of nanoparticles on the differentiation capabilities of rdMSC to osteoblasts. In addition the production of mineralized matrix has been shown by von-Kossa stained samples. No influence of the nanoparticles on the ALP per cell has been detected. Additionally, for all experiments, results are strongly influenced by a large donor-to-donor variability of the four different rdMSC samples. To summarize, while featuring a good cellular uptake, PEI-5k-Mal-B induces only minimal adverse effects and features clearly superior biocompatibility compared to the larger PEI-25k-Mal-B.

No MeSH data available.


Related in: MedlinePlus