Fig. 3

Proteoglycan protein expression in co-cultures. Alcian blue staining of a representative donor sample is shown (donor #15) (a–c). a An overview of a co-culture including one implanted spheroid (S) and (b) higher magnification of the implanted spheroid, surrounded by GAG positive ECM fibers (EF) (c) GAG containing regenerative tissue (RT) and multilayer (ML) at the surface. Alcian blue staining stains GAGs blue, and nuclear fast red stained the nuclei red. (d–f) Immunohistochemical detection of aggrecan protein expression shown as red staining of representative donor samples. ACAN protein expression in donor #8 (c) showing a low expression in implanted spheroids (S) and a high expression in ECM fibers (EF) connected to the native cartilage (NC). d ACAN protein expression in the ML of the regenerative tissue in donor #8, as well as in the ECM fibers. Single cells (arrowheads) were often detected within the ECM fibers. e IgG isotype control. Sections were counterstained with haematoxylin, staining the nuclei blue. Scale bar of a: 500 µm; b–f, 100 µm; EF ECM fibers, ML multilayer, NC native cartilage, RT regenerative tissue, S spheroid, SB subchondral bone