Fig 5. H2S promotes osteogenic differentiation in 3D spheroids.
HDFs were cultured in 3D in the absence and presence of 50 μM DATS for 6 days. The cells were then cultured in DMEM and osteogenic conditions for up to 21 days. A. Alizarin Red staining. B. Alkaline phosphatase staining. C. Densitometric analysis of Alizarin staining. D. mRNA expression levels of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (OCN) also referred to as bone γ-carboxyglutamic acid-containing protein or BGLAP, and RUNX2, and expression of were evaluated in 2D and 3D cultures maintained in DMEM and culture condition allowing osteogenic differentiation. Expression levels were normalized to the expression level in monolayer (2D) cells cultured in DMEM. E. HDFs were cultured in 3D in the absence and presence of 50 μM DATS or 100 μM of NaHS for 6 days and then subjected to osteogenic differentiation. qPCR analysis of RUNX2 gene expression normalized to the expression level in 2D cultured cells. Statistical significance is shown as * (p≤0.05), ** (p ≤0.005), and *** (p ≤0.0005) in 3D as compared to 2D cultured cells.