Abstract
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.
Highlights
NELL-1 is a potent growth factor, reported to target cells committed to the osteochondral lineage [1]
The purpose of this study is to reveal the effects of NELL-1 on the morphology of spheroids, maintenance of cell viability, and enhancement of osteogenic differentiation using threedimensional cultures of stem cells
The activity of alkaline phosphatase and anthraquinone dye staining were used to evaluate the osteogenic differentiation of stem cell spheroids on Days 7 and 14 [21]
Summary
NELL-1 is a potent growth factor, reported to target cells committed to the osteochondral lineage [1]. NELL-1 is reported to induce odontoblast differentiation and pulp capping with enhanced formation of reparative dentin and reduced inflammatory cell responses, suggesting it as a positive regulator for pulp repair [7]. Infusion of mesenchymal stem cells improved bone performance as a result of stem cell differentiation into osteoblasts [12]. NELL-1 significantly induced mesenchymal stem cell migration [15]. Spheroids are shown to facilitate cell-to-cell interaction and to produce an increased osteogenic potential [17]. The purpose of this study is to reveal the effects of NELL-1 on the morphology of spheroids, maintenance of cell viability, and enhancement of osteogenic differentiation using threedimensional cultures of stem cells
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