Establishment of organ culture model of disc degeneration. (A) Schematic showing disc organ culture setup. Culturing discs under low oxygen partial pressure (pO2) in hyperosmolar, nutritionally limiting media and exposure to proinflammatory cytokines mimic molecular changes characteristic of early disc degeneration. NP, nucleus pulposus; AF, annulus fibrosus; FBS, fetal bovine serum. (B,C) Measurement of luciferase diffusion in nucleus pulposus (NP) tissue after 24 hours. Both Firefly (61 kDa) and Renilla (38 KDa) luciferase diffuse in the tissue in a concentration-dependent fashion. Results are shown as the mean ± standard error for three independent experiments performed in triplicate. *P <0.05. (D) Histological sections were stained for apoptotic cells. Tumor necrosis factor-alpha (TNF-α)-treated and interleukin-1beta (IL-1β)-treated intervertebral discs demonstrated significantly higher numbers of apoptosis-positive cells (d) than the untreated control group (b) after 3 days in culture (DAPI: a and c). Ctr., control group; DAPI, 4'-6-diamidino-2-phenylindole; Exp., experimental group; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. (E) Histological analysis of disc after treatment. Sections are stained with Alcian blue and hematoxylin-and-eosin counter staining and photographed with a low-magnification (4×) lens (a). Panels (b) and (c) were examined under high magnification (20×). Aberrant cellular hypertrophy and matrix degradation of NP tissue in treated disc were observed after 3 and 10 days. Representative findings of three separate experiments (n = 3 discs/group per experiment) are shown.