EAO‐368/PO‐BR‐005 | The effects of vitamin D on osteogenic differentiation of the stem cell spheroids

Background and objectives: NELL-1 is a competent growth factor and it reported to target cells committed to the osteochondral lineage. The secreted, osteoinductive glycoproteins are reported to rheostatically control skeletal ossification. This study was performed to determine the effects of NELL-1 on spheroid morphology and cell viability and the promotion of osteogenic differentiation of stem cell spheroids. Materials and Methods: Cultures of stem cell spheroids of gingiva-derived stem cells were grown in the presence of NELL-1 at concentrations of 1, 10, 100, and 500 ng/mL. Evaluations of cell morphology were performed using a microscope, and cell viability was assessed using a two-color assay and Cell Counting Kit-8. Evaluation of the activity of alkaline phosphatase and calcium deposition assays involved anthraquinone dye assay to determine the level of osteogenic differentiation of cell spheroids treated with NELL-1. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expressions of RUNX2, BSP, OCN, COL1A1, and β-actin mRNAs. Results: The applied stem cells produced well-formed spheroids, and the addition of NELL-1 at tested concentrations did not show any apparent changes in spheroid shape. There were no significant changes in diameter with addition of NELL-1 at 0, 1, 10, 100, and 500 ng/mL concentrations. The quantitative cell viability results derived on Days 1, 3, and 7 did not show significant disparities among groups (p > 0.05). There was statistically higher alkaline phosphatase activity in the 10 ng/mL group compared with the unloaded control on Day 7 (p < 0.05). A significant increase in anthraquinone dye staining was observed with the addition of NELL-1, and the highest value was noted at 10 ng/mL (p < 0.05). qPCR results demonstrated that the mRNA expression levels of RUNX2 and BSP were significantly increased when NELL-1 was added to the culture. Conclusions: Based on these findings, we conclude that NELL-1 can be applied for increased osteogenic differentiation of stem cell spheroids.

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Vitamin D Enhanced the Osteogenic Differentiation of Cell Spheroids Composed of Bone Marrow Stem Cells

Medicina ◽

10.3390/medicina57111271 ◽

2021 ◽

Vol 57 (11) ◽

pp. 1271

Author(s):

Hyun-Jin Lee ◽

Young-Min Song ◽

Seunghoon Baek ◽

Yoon-Hee Park ◽

Jun-Beom Park

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Vitamin D ◽

Polymerase Chain Reaction ◽

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Cellular Viability ◽

Chain Reaction ◽

Cell Spheroids ◽

Polymerase Chain

Background and Objectives: Vitamin D is a bone modulator widely used in regenerative medicine. This study aimed to analyze the effects of vitamin D on the osteogenic differentiation and mineralization of human mesenchymal stem cells. Materials and Methods: Spheroids were fabricated using human bone marrow-derived stem cells, and were cultured in the presence of vitamin D at concentrations of 0, 0.1, 1, 10, and 100 nM. Stem cell spheroids were fabricated and the morphological evaluation was conducted on days 1, 3, 7 and 14. Determination of qualitative cellular viability was performed with Live/Dead Kit assay on days 1 and 7. Quantitative cellular viability was evaluated with Cell Counting Kit-8 on days 1, 3, 7, and 14. To analyze the osteogenic differentiation of cell spheroids, alkaline phosphatase activity assays were performed with commercially available kit on days 7 and 14. Real-time polymerase chain reaction was used to determine the expression levels of RUNX2, BSP, OCN, and COL1A1 on days 7 and 14. Results: The stem cells produced well-formed spheroids, and addition of vitamin D did not result in any noticeable changes in the shape. The addition of vitamin D did not significantly change the diameter of the spheroids at 0, 0.1, 1, 10, or 100 nM concentrations. Quantitative cell viability results from days 1, 3, 7 and 14 showed no significant difference between groups (p > 0.05). There was significantly higher alkaline phosphatase activity in the 0.1 nM group when compared with the control group on day 14 (p < 0.05). Real-time polymerase chain reaction results demonstrated that the mRNA expression levels of RUNX2, OCN, and COL1A1 were significantly increased when vitamin D was added to the culture. Conclusions: Based on these findings, we concluded that vitamin D could be applied to the increased osteogenicity of stem cell spheroids.

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