ABSTRACT
The aim of the present study was to evaluate the morphology, cellular viability and stem cell marker expression of three-dimensional cultures of bone marrow and gingiva-derived stem cells in different ratios. Stem cell spheroids were made with bone marrow and gingiva-derived stem cells using ratios of 6:0 (Group 1), 4:2 (Group 2), 3:3 (Group 3), 2:4 (Group 4) and 0:6 (Group 5), respectively. The viability of cell spheroids was analyzed using a Live/Dead kit assay and a Cell Counting Kit-8 assay. Total RNA extraction and reverse transcription-quantitative PCR were performed to detect the mRNA expression levels of Nanog and ß-actin in each group. Stem cell spheroids were well formed in silicone elastomer-based concave microwells with different ratios of bone marrow and gingiva-derived stem cells. The shape of the spheroids and their viability were maintained throughout the entirety of the experimental procedure. Statistically significant increases in spheroid diameters were noted in Groups 4 and 5 on day 1 when compared with Group 1 on day 1. There was a significant increase in the cell viability values seen in Group 3 on day 1 when compared with Group 1 on day 1. Highest levels of Nanog expression was seen in Group 3 on day 10, but the increase was not significant when compared with Group 1 on day 1. Co-culturing with higher ratios of gingiva-derived stem cells produced stem cell spheroids with larger diameters and increased cellular viability. This co-culture technique may be used in stem cell therapy with allogenic stem cell transplantation.
ABSTRACT
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.
ABSTRACT
Lovastatin is a cholesterol-lowering agent that also has effects of cell proliferation and apoptosis. The present study was performed to evaluate the effects of lovastatin on the proliferation and osteogenic differentiation of three-dimensional cell spheroids formed from human gingiva-derived stem cells (GDSCs) using concave microwells. GDSCs were plated on polydimethylsiloxane-based concave micromolds and grown in the presence of lovastatin at concentrations of 0, 2 and 6 µM. The morphology of the cells was viewed under an inverted microscope, and cell viability was determined with Cell Counting kit-8 on days 2, 7 and 14. Alkaline phosphatase activity assays were performed to evaluate the osteogenic differentiation on days 2 and 8. Alizarin red-S staining was also used to assess the mineralization of the stem cell spheroids at day 14. The results confirmed that GDSCs formed spheroids in concave microwells. No significant changes were noted with longer incubation time, and no significant differences in cell viability were noted between the three lovastatin groups at each time point. Higher osteogenic differentiation was observed in the 2 µM group when compared with the control. Mineralized extracellular deposits were visible after Alizarin red-S staining, and higher mineralization was noted in the 2 and 6 µM lovastatin groups when compared with the 0 µM control. The relative mineralization values of the 0, 2 and 6 µM groups on day 14 were 39.0±9.6, 69.3±6.0 and 60.9±7.5, respectively. This study demonstrated that the application of lovastatin enhanced the osteogenic differentiation of cell spheroids formed from GDSCs. This suggests that combinations of lovastatin and stem cell spheroids may have the potential for use in tissue engineering.
ABSTRACT
Fibroblast growth factor-2 (FGF-2) is reported to have various functions and is considered a key human mesenchymal stem cell mitogen, often supplemented to increase human mesenchymal stem cell growth rates. The purpose of this study was to evaluate the effects of FGF-2 on cellular viability and osteogenic differentiation using three-dimensional cell spheroids of stem cells. Three-dimensional cell spheroids were fabricated using concave silicon elastomer-based microwells in the presence of FGF-2 at concentrations of 0, 30, 60 and 90 ng/ml. Qualitative cellular viability was determined with a confocal microscope, and quantitative cellular viability was evaluated using a Cell Counting Kit-8 assay. Alkaline phosphatase activity and Alizarin Red S staining were used to assess osteogenic differentiation. Spheroids were well formed in silicon elastomer-based concave microwells on Day 1. The average spheroid diameters at Day 1 for FGF-2 at 0, 30, 60 and 90 ng/ml were 202.2±3.0, 206.6±22.6, 208.8±6.8 and 196.6±26.7 µm, respectively (P>0.05). The majority of the cells in the cell spheroids emitted green fluorescence. The relative Cell Counting Kit-8 assay values for FGF-2 at 0, 30, 60 and 90 ng/ml at Day 1 were 100.0±5.5, 101.8±8.8, 99.2±4.8 and 103.4±9.6% (P>0.05). The addition of FGF-2 at 60 ng/ml concentration produced the highest value for alkaline phosphatase activity. Mineralized extracellular deposits were evenly observed in each group, and the highest value was identified for FGF-2 groups at 60 ng/ml concentration for Alizarin Red S staining. Based on these findings, it was concluded that FGF-2 may increase alkaline phosphatase activity or Alizarin Red S staining, and further studies are needed to fully elucidate the mechanisms of FGF-2.
ABSTRACT
The aim of the current study was to evaluate cell viability and osteogenic differentiation potential in cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells cultured in concave microwells. Cell spheroids were established from bone marrow and gingiva-derived stem cells in ratios of 6:0 (Group 1), 2:1 (Group 2), 3:3 (Group 3), 1:2 (Group 4), and 0:6 (Group 5). On days 3 and 5, the viability of the cell spheroids was qualitatively analyzed using a calcein acetoxymethyl ester working solution and an ethidium homodimer-1 live/dead assay. On days 1, 3, 5 and 7, a quantitative cell viability analysis was performed using a Cell Counting Kit-8. Alkaline phosphatase activity assays were performed using a commercially available kit on day 7 to assess osteogenic differentiation. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to evaluate runt-related transcription factor 2 (Runx2) and osteocalcin expression. The ratio of gingiva-derived to bone marrow stem cells did not affect the stem cell spheroid morphology. No significant changes in cell viability were noted among the different groups following incubation for 7 days. A consistent alkaline phosphatase activity was measured in co-cultured gingiva-derived and bone marrow stem cell spheroids of varying compositions. Runx2 and osteocalcin expression was increased when co-cultured compared with pure gingiva-derived or bone marrow stem cells. In conclusion, stem cell spheroids established by co-culturing maintained morphology, viability and a high osteogenic differentiation potential during the experimental period of 7 days. These spheroids containing human gingiva-derived and bone marrow stem cells may enhance the osteogenic differentiation potential. The use of multicell spheroids may be a simple and effective strategy for improving stem cell therapy.
ABSTRACT
PURPOSE: This study was performed to fabricate stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells and to evaluate their viability and osteogenic differentiation potential. MATERIALS AND METHODS: Gingiva-derived stem cells were isolated, and stem cells and endothelial cells with a total of 6 × 10 cells were seeded into concave micromolds with different ratios of 6:0 (group 1), 4:2 (group 2), 3:3 (group 3), and 2:4 (group 4). RESULTS: Gingiva-derived stem cells and/or endothelia cells formed spheroids in concave microwells. There was a decreasing trend in the diameter of spheroids with increasing amounts of endothelial cells, but there were no statistically significant differences between the groups. The secretion of vascular endothelial growth factor from the spheroids was noted. The results of the alkaline phosphatase activity assays showed significantly higher values for groups 2, 3, and 4 when compared with the value of group 1. CONCLUSIONS: Conclusively, stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells using concave microwells enhanced osteogenic differentiation potential, and multicell spheroid-based cell delivery could be a simple and effective strategy for improving stem-cell therapy.
