Figure 4.

Purification method changes the effect of exosomes from BM-MSCs on the eAC expression of cartilage- and OA-associated proteins. CMs from BM-MSCs (P3) were harvested and exosomes were isolated using either the MAC or the SEC method. Unconditioned medium (M3D) was aliquoted, supplemented with fresh exosomes and stored at −80 °C. A chondrogenic control was included by supplementing M3D with BMP2 (50 ng/mL). CM3D corresponds to BM-MSC culture medium conditioned for 24 h. The 1X and 2X concentrations refer to protein concentrations of exosome solution related to 1 mL of CM3D. eACs (P2) were seeded in collagen sponges at 800,000 cells/sponge and then cultured under hypoxic atmosphere with the different media for 14 days. Then, sponges were harvested, washed twice with PBS, and stored at −80 °C. The total proteins were extracted using a dedicated buffer supplemented with protease inhibitors and Western blots were carried out to evaluate protein levels, as shown in the representative blots. The same protein targets are presented in subfigures (A,C), differing from blots presented in subfigure (B). The D0 condition corresponds to eACs cultured in monolayer until P3. Arrows indicate type X collagen bands. Experiments were reproduced with different strains of eACs and BM-MSCs (n = 4). BM-MSCs, bone marrow-mesenchymal stromal cells; CM, conditioned medium; eACs, equine articular chondrocytes; M3D, control medium with 2% FBS; P2, passage 2; P3, passage 3.