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. 2024 Jan 2;33:09636897231221878. doi: 10.1177/09636897231221878

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© The Author(s) 2024

This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Figure 6. — Detection of osteocyte-related gene expressions of adult and infant BMSCs during differentiation using OIM. (A) The expression of ALP and (B) OPN genes in adult and infant BMSCs during osteogenic differentiation from day 7 to day 21 was detected using RT-qPCR. The expression values of each gene were normalized to the expression of GAPDH. Undifferentiated cells were used as a control to compare the gene expression of adult and infant BMSCs after differentiation, showing the relative fold change. (C) Undifferentiated adult and infant BMSCs were stained with ARS as a control. The 21-day osteogenic differentiated adult and infant BMSCs were confirmed using ARS staining and observed under a Nikon eclipse TS100 microscope (magnification × 200, scale bar = 25 μm). (D) The ARS stain was extracted from the stained cells, and the optical density (OD) was measured at a wavelength of 550 nm. The OD values of the differentiated cells were normalized to the control values set as 1 (not shown in the figure), demonstrating the relative fold changes in comparison to the control. The mean ± standard error of the mean (SEM) calculated from three experimental replicates within both the infant and adult groups. Statistical significance of comparing adult and infant BMSCs in osteogenic differentiation was determined using the Mann–Whitney U test. BMSC: bone marrow–derived mesenchymal stem cell; ALP: alkaline phosphatase; OPN: osteopontin; OIM: osteogenic induction medium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ARS: Alizarin Red S; RT-qPCR: real-time quantitative polymerase chain reaction. The significance levels are indicated by *P < 0.05; ***P < 0.001.