FIGURE 2.

H-Exo were superior to L-Exo in promoting osteogenic differentiation in the TCs. (A) Exosomes obtained from H-SCCs and L-SCCs were identified by TEM. (B) Size distribution was assessed using a ZetaView nanoparticle tracking analyzer. (C) The expression of the exosomal marker proteins CD9, CD81, and TSG101 was tested by western blot. (D) Osteogenic inducing fluid containing 20 μg/ml exosomes was used, and the expression of the osteogenic differentiation-related genes in unstimulated TCs, or TCs stimulated by L-Exo or H-Exo, was measured by RT-qPCR after 7 days of osteogenic induction (n = 3). (E) Osteogenic inducing fluid containing 20 μg/ml exosomes was used, and Alizarin red staining of unstimulated TCs, or TCs stimulated by L-Exo or H-Exo, was performed after 28 days of osteogenic induction (n = 3). (F) The collected supernatants from the culture of DMSO- or GW4869-treated SCCs were mixed with equal volumes of twofold concentrated osteogenic induction medium to stimulate the TCs, and the expression levels of the osteogenic differentiation-related genes in the TCs were determined by RT-qPCR after 7 days of osteogenic induction (n = 3 or 4). (G) The collected supernatants from the culture of DMSO- or GW4869-treated SCCs were mixed with equal volumes of twofold concentrated osteogenic induction medium to stimulate the TCs, and Alizarin red staining of the TCs was performed after 28 days of osteogenic induction (n = 3). L-Exo, exosomes secreted by L-SCCs; L-SCCs + DMSO, TCs stimulated by the culture of DMSO-treated L-SCCs; L-SCCs + GW4869, TCs stimulated by the culture of GW4869-treated L-SCCs; H-Exo, exosomes secreted by H-SCCs; H-SCCs + DMSO, TCs stimulated by the culture of DMSO-treated H-SCCs; H-SCCs + GW4869, TCs stimulated by the culture of GW4869-treated H-SCCs; TCs, target cells; TEM, transmission electron microscopy. Scale bar, 100 nm. *P < 0.05; **P < 0.01; ***P < 0.001.