Figure 5.

Avitene^TM microfibrillar collagen scaffolds reconstituted with lymph node-mesenchymal stromal and Hodgkin lymphoma cells as 3D culture model to test ADAM10 inhibitors. Avitene^TM scaffolds were cultured with 2x10⁵ LN-MSC16412 for 3 days, followed by 4x10⁵ L428 (A to C and D to E left histograms) or L540 cells (D to E right histograms) for further 2 days, before addition of 10 mM LT4 or MN8 for 72 hours (h) or 96 h. (A) 4 mm sections of repopulated Avitene^TM scaffolds stained with anti-TGII polyclonal antiserum followed by Biot-GAR, HRP-Av and developed with DAB; (B) sections stained with anti-CD30 monoclonal antibody (mAb) followed by Biot-GAM, HRP-Av and developed with DAB. Inset in the left pictures: images of the whole repopulated scaffold. Inset in the right pictures: negative control (Nil) with Biot-GAR(A) or Biot-GAM (B) alone. Slides were counterstained with hematoxylin and analyzed under a Leica DM MB2 microscope (left: 20x enlargement, right: 40x enlargement). (C) Scanning electron microscopy (SEM) images of Avitene^TM scaffolds, repopulated with LN-MSC16412 and L428 cells (arrows). Magnifications and scale bar are reported in each panel. Inset in the left picture: the whole scaffold with a white square indicating the area enlarged. (D and E) Soluble CD30 (D) or TNFa (E) content (pg/mL/10⁶ cells), measured by specific enzyme-linked immunosorbant assay, in the supernatant (SN) recovered after 72 h or 96 h from addition of ADAM10 inhibitors to the scaffolds repopulated with LN-MSC16412 and L428 (left histograms) orL540 (right histograms). Results are the mean the mean ± standard deviation of quadruplicates from three independent experiments.**P<0.005 vs. nil; ***P<0.001 vs. nil.