Cell layers were lysed by ultrasonication at 40V for 15 seconds/well (VCX 130; Vibra-Cell, Newtown, CT). The QuantiFluor* dsDNA system (Promega, Madison, WI) was used to determine total DNA content by fluorescence. Alkaline phosphatase activity was measured in the cell layer lysates by the release of para-nitrophenol from para-nitrophenyl phosphate (pH 10.2) and normalized to time and protein content measured by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA).
Enzyme-linked immunosorbent assays were used to determine the levels of osteogenic factors in the conditioned media. Osteocalcin (OCN; Thermo Fisher), osteoprotegerin (OPG; R&D Systems, Inc.), osteopontin (OPN; R&D Systems, Inc.), vascular endothelial growth factor A (VEGF-A; R&D Systems, Inc.), BMP2 (R&D Systems, Inc.), bone morphogenetic protein 4 (BMP4; R&D Systems, Inc.), interleukin 6 (IL6; R&D Systems, Inc.), and interleukin 10 (IL10; R&D Systems, Inc.) were quantified according to the manufacturer’s protocol.
Enzyme-linked immunosorbent assays were used to determine the levels of osteogenic factors in the conditioned media. Osteocalcin (OCN; Thermo Fisher), osteoprotegerin (OPG; R&D Systems, Inc.), osteopontin (OPN; R&D Systems, Inc.), vascular endothelial growth factor A (VEGF-A; R&D Systems, Inc.), BMP2 (R&D Systems, Inc.), bone morphogenetic protein 4 (BMP4; R&D Systems, Inc.), interleukin 6 (IL6; R&D Systems, Inc.), and interleukin 10 (IL10; R&D Systems, Inc.) were quantified according to the manufacturer’s protocol.