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. Tissue non-specific 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 (Thermo Fisher), osteoprotegerin (R&D Systems, Inc., Minneapolis, MN), vascular endothelial growth factor A (R&D Systems, Inc.), bone morphogenetic protein 2 (Pepro Tech, Rocky Hill, NJ), interleukin 6 (R&D Systems, Inc.), and interleukin 10 (R&D Systems, Inc.) were quantified according to manufacturer’s protocol.
Enzyme-linked immunosorbent assays were used to determine the levels of osteogenic factors in the conditioned media. Osteocalcin (Thermo Fisher), osteoprotegerin (R&D Systems, Inc., Minneapolis, MN), vascular endothelial growth factor A (R&D Systems, Inc.), bone morphogenetic protein 2 (Pepro Tech, Rocky Hill, NJ), interleukin 6 (R&D Systems, Inc.), and interleukin 10 (R&D Systems, Inc.) were quantified according to manufacturer’s protocol.