The proteoglycan metabolism of articular cartilage in joint-scale culture

Abstract

Understanding and controlling chondrocyte and cartilage metabolism in osteochondral tissues may facilitate ex vivo maintenance and application, both for allografts and tissue-engineered grafts. The hypothesis of this study was that maintenance of chondrocyte viability and matrix content and release of sulfated glycosaminoglycan (sGAG) in the articular cartilage of joint-scale osteochondral fragments are temperature and metabolism dependent. The aims were to assess, for adult goat joints, the effects of incubation temperature (37 degrees C vs. 4 degrees C) on cartilage chondrocyte viability and tissue matrix content and mechanical function, and the effects of temperature and cellular biosynthesis on sGAG release. Chondrocyte viability was maintained with 37 degrees C incubation for 28 days, but decreased by approximately 30% with 4 degrees C incubation. Concomitantly, with 37 degrees C incubation, cartilage sGAG was depleted by approximately 52% with the lost sGAG predominantly unable to aggregate with hyaluronan, whereas collagen content, tissue thickness, and tissue stiffness were maintained. The depletion of sGAG was diminished by slowing metabolism, with 4 degrees C decreasing release by approximately 79% compared with 37 degrees C incubation, and cycloheximide inhibition of cell metabolism at 37 degrees C decreasing release by approximately 47%. These results indicate that the articular cartilage of joint-scale grafts have enhanced chondrocyte viability with incubation at 37 degrees C, but may need anabolic stimuli or catabolic inhibitors to maintain sGAG content.

FIG. 1.

Schematic diagrams of (A) experimental groups, (B) joint-scale bioreactor setup, and (C) core isolation locations from the distal humerus. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Osteochondral cores were analyzed for chondrocyte viability by Live/Dead^® fluorescence staining in the en face (A–D) and vertical (E–H) profiles. Representative images of cores from fresh control (A, E), 12 days at 37°C (B, F), 28 days at 37°C (C, G), and 28 days at 4°C (D, H) are shown. Scale bar: 100 μm. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Fluorescent Live/Dead images were analyzed for percentage viable chondrocytes in the en face profile (A) and the middle (B) and deep (C) zones of the vertical profile from fresh control, 37°C at 12 days, 37°C at 28 days, and 4°C at 28 days. ^#Significantly less than all other bars (p < 0.05); ^##significantly less than fresh control and 37°C at 28 days groups (p < 0.05).

FIG. 4.

Osteochondral cores from fresh control, 37°C at 12 days, 37°C at 28 days, and 4°C at 28 days were analyzed for thickness (A), indentation stiffness (B), and sGAG content (C). **p < 0.01 and ***p < 0.001. sGAG, sulfated glycosaminoglycan.

FIG. 5.

Sections of osteochondral cores from fresh control (A), 28 days 37°C (B), and 28 days 4°C (C) were prepared and stained with Toluidine blue. Scale bar: 200 μm. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Conditioned medium samples collected every 3.5 days were analyzed for sGAG content and normalized to joint surface area. Joints were incubated with (filled) or without (open) 0.5 mM cycloheximide (CX) at 4°C, 37°C, or for 7 days at 4°C and then 7 days at 37°C (4/37°C) (A). Within each time point, letters (a, b, and c) designate significantly different groups (p < 0.05), with a shared letter indicating no difference. *Initial time point not different from second point, but different from the remaining time points (p < 0.05), **Initial time point different from the remaining time points (p < 0.05). Average sGAG release for each group over 0–7 days (B) and 7–14 days (C) was normalized to the sGAG release from the 37–CX group from 0 to 7 days to emphasize differences due to CX and temperature.

FIG. 7.

Percentage of total sGAG content (sGAG_tissue + sGAG_media) detected in the core digests (dark gray) and cumulative amount in the medium summed over the incubation period (light gray). Incubation time, temperature (4/37 indicates 1 week at 4°C and then 1 week at 37°C), and the presence or absence of 0.5 mM CX are indicated. **p < 0.01; ***p < 0.001; ^#different than all other groups, p < 0.001; ^##different than all other groups (p < 0.001–0.05) except 14 days at 4/37 with CX (p = 0.58).

FIG. 8.

Fresh extract controls (lanes 1–3) and spent medium (day 3.5 at 37°C, lanes 4–6) samples were separated on a 1% agarose gel and stained with Alcian blue.