Figure 1.

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.