The effects of simvastatin on cellular viability, stemness
and osteogenic differentiation using 3-dimensional
cultures of stem cells and osteoblast-like cells

Lee, Hyunjin; Na, Chae-Bin; Park, Jun‐Beom

doi:10.17219/acem/94162

Cited by 17 publications

(17 citation statements)

References 25 publications

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“…The dye was removed and the spheroids were washed three times with deionized water. The quantification of the bound dyes was performed afterwards by adding 10% cetylpyridinium chloride for 15 min at ambient temperature, and we performed spectrophotometric quantification at 560 nm [ 9 ].…”

Section: Methodsmentioning

confidence: 99%

“…Recently, scaffold-free techniques have been of great interest because this technique allows the various types of cells, including stem cells, to form cell aggregates called spheroids [ 6 , 7 ]. Spheroids can be fabricated in various sizes and shapes, and the reproducible size/shape of spheroids can be obtained [ 8 , 9 ]. Spheroids can secrete growth factors and display differential nutrient and gas availability [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

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The Effects of Morinda citrifolia (Noni) on the Cellular Viability and Osteogenesis of Stem Cell Spheroids

Min

Park

2020

Medicina

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Background and objectives: Morinda citrifolia (Noni) has been widely used in herbal remedies to treat and prevent various kinds of diseases. We conducted this study to evaluate the effects of Noni extract on the maintenance of morphology, the improvement of cellular viability, and the enhancement of osteogenesis of stem cell spheroids. Materials and Methods: We cultured stem cell spheroids made with gingiva-derived stem cells in the presence of Noni extract at concentrations of 10, 100 and 200 ng/mL. We performed analysis of the cell morphology and changes in the cellular viability. We conducted alkaline phosphatase activity assays using a kit, and mineralization assays using an anthraquinone dye to evaluate the osteogenesis of stem cell spheroids with the addition of Noni extract. Results: The applied cells formed spheroids well, and the addition of Noni at 10, 100 and 200 ng/mL concentrations did not produce significant morphological changes. The quantitative values for cellular viability on Day 3 showed that the absorbance values at 450 nm were 0.314 ± 0.013, 0.318 ± 0.008, 0.304 ± 0.000 and 0.300 ± 0.011 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively. The results of alkaline phosphatase activity with absorbance values at 405 nm were 0.189 ± 0.019, 0.174 ± 0.023, 0.192 ± 0.014 and 0.210 ± 0.062 for Noni at 0, 10, 100 and 200 ng/mL concentrations, respectively, on Day 4. There were significantly higher values of Alizarin Red S staining for Noni in the 10, 100 and 200 ng/mL groups, with the highest value at 100 ng/mL when compared with the unloaded control on Day 14. Conclusions: Based on these findings, we concluded that Noni extract might be applied for the enhanced osteogenic differentiation of stem cell spheroids.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Effects of Morinda citrifolia (Noni) on the Cellular Viability and Osteogenesis of Stem Cell Spheroids

Min

Park

2020

Medicina

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“…The expressions of various genes were tested to evaluate the osteogenic differentiation including Runx 2 and BGLAP [17,30]. Runx2 and BGLAP are widely used biomarkers for osteogenic differentiation [30].…”

Section: Discussionmentioning

confidence: 99%

“…We harvested the cells on Day 7 and isolated total RNA using purification kit (Thermo Fisher Scientific, Inc.), following the manufacturer's instructions [17]. RNA quality was assessed with a bioanalyzer (Agilent 2100, Agilent Technologies) using the kit (RNA 6000 Nano Chip; Agilent Technologies, Amstelveen, The Netherlands), and the quantity was performed using the spectrophotometer (ND-2000, Thermo Fisher Scientific, Inc.) with a ratio of absorbance at 260 nm and 280 nm spectrophotometrically.…”

Section: Mrna Quantification By Real-time Polymerase Chain Reactionmentioning

confidence: 99%

The Role of Insulin-Like Growth Factor-2 on the Cellular Viability and Differentiation to the Osteogenic Lineage and Mineralization of Stem Cells Cultured on Deproteinized Bovine Bone Mineral

Lee

Min

Park³

et al. 2020

Applied Sciences

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Insulin-like growth factors (IGFs) plays various roles, including differentiation and mitogenesis, and IGFs are reported to regulate the bone growth and maintenance. This study was performed to analyze the enhancing effects of IGF-2 on osteogenic differentiation and the mineralization of stem cells cultured on deproteinized bovine bone mineral. Stem cell loaded bone graft material was cultured in the presence of the IGF-2 at final concentrations of 10 and 100 ng/mL and the morphology of the cells was observed on Days 1, 3, and 7. The commercially available, two-color assay based on plasma membrane integrity and esterase activity was also used for qualitative analyses on Days 1, 3, and 7. The level of alkaline phosphatase activity and anthraquinone dye assay were used to evaluate osteogenic differentiation on Days 7 and 14. Real-time polymerase chain reaction was applied in order to identify the mRNA expression of BGLAP, Runx2, and β-catenin. The stem cells were well-attached with fibroblast morphology and most of the stem cells produced a high intensity of green fluorescence, indicating that there were live cells on Day 1. The relative cellular viability assay values for IGF-2 groups at 0, 10, and 100 ng/mL on Day 1 were 0.419 ± 0.015, 0.427 ± 0.013, and 0.500 ± 0.030, respectively (p < 0.05). The absorbance values at 405 nm for alkaline phosphatase activity on Day 7 for IGF-2 at 0, 10, and 100 ng/mL were 2.112 ± 0.152, 1.897 ± 0.144, and 2.067 ± 0.128, respectively (p > 0.05). The mineralization assay results at Day 7 showed significantly higher values for IGF-2 groups at 10 and 100 ng/mL concentration when compared to the control (p < 0.05). The application of IGF-2 groups of 10 and 100 ng/mL produced a significant increase of BGLAP. Conclusively, this study indicates that the use of IGF-2 on stem cell loaded bone graft increased cellular viability, Alizarin red staining, and BGLAP expression of stem cells. This report suggests the combined approach of stem cells and IGF-2 with scaffold may have synergistic effects on osteogenesis.

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“…Total RNA was extracted from cell spheroids using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at day 8 (21,22). SuperScript II RTase (Invitrogen; Thermo Fisher Scientific, Inc.) was used for RT with total RNA according to the manufacturer's instructions at 42˚C for 50 min.…”

Section: Mrna Quantification By Reverse Transcription-quantitative Pcmentioning

confidence: 99%

Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression

et al. 2020

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Fibroblast growth factors (FGFs) are growth factors that were initially identified as proteins that stimulate fibroblast proliferation. The aim of the present study was to examine the effects of FGF-4 on the morphology, cellular viability and osteogenic differentiation of stem cell spheroids. Stem cell spheroids were generated using concave microwells in the presence of FGF-4 at concentrations of 0, 50, 100 and 200 ng/ml. Cellular viability was qualitatively assessed by a fluorometric live/dead assay using a microscope and quantitatively determined by using Cell Counting Kit-8. Furthermore, alkaline phosphatase activity and calcium deposition were determined to assess osteogenic differentiation. Reverse transcription-quantitative PCR (RT-qPCR) was performed to evaluate the mRNA expression levels of Runt-related transcription factor 2 (RUNX2) and bone γ-carboxyglutamate protein (BGLAP). Spheroidal shapes were achieved in the microwells on day 1 and a significant increase in the spheroid diameter was observed in the 200 ng/ml FGF-4 group compared with the control group on day 1 (P<0.05). The results regarding viability using Cell Counting Kit-8 in the presence of FGF-4 at 50, 100 and 200 ng/ml at day 1 were 98.0±2.5, 106.2±17.6 and 99.5±6.0%, respectively, when normalized to the control group (P>0.05). Furthermore, the alkaline phosphatase activity was significantly elevated in the 200 ng/ml group, when compared with the control group. The RT-qPCR results demonstrated that the mRNA expression levels of RUNX2 and BGLAP were significantly increased at 200 ng/ml. Therefore, the present results suggested that the application of FGF-4 maintained cellular viability while enhancing the osteogenic differentiation of stem cell spheroids, at least partially by regulating RUNX2 and BGLAP expression levels.

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The effects of simvastatin on cellular viability, stemness and osteogenic differentiation using 3-dimensional cultures of stem cells and osteoblast-like cells

Cited by 17 publications

References 25 publications

The Effects of Morinda citrifolia (Noni) on the Cellular Viability and Osteogenesis of Stem Cell Spheroids

The Effects of Morinda citrifolia (Noni) on the Cellular Viability and Osteogenesis of Stem Cell Spheroids

The Role of Insulin-Like Growth Factor-2 on the Cellular Viability and Differentiation to the Osteogenic Lineage and Mineralization of Stem Cells Cultured on Deproteinized Bovine Bone Mineral

Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression

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