Menisci protect chondrocytes from load-induced injury

Abstract

Menisci in the knee joint are thought to provide stability, increased contact area, decreased contact pressures, and offer protection to the underlying articular cartilage and bone during joint loading. Meniscal loss or injury is typically accompanied by degenerative changes in the knee, leading to an increased risk for osteoarthritis in animals including humans. However, the detailed mechanisms underlying joint degeneration and the development of osteoarthritis remain largely unknown, and the acute effects of meniscal loss have not been studied systematically. We developed a microscopy-based system to study microscale joint mechanics in living mice loaded by controlled muscular contractions. Here, we show how meniscal loss is associated with rapid chondrocyte death (necrosis) in articular cartilage within hours of injury, and how intact menisci protect chondrocytes in vivo in the presence of intense muscle-based joint loading and/or injury to the articular cartilage. Our findings suggest that loading the knee after meniscal loss is associated with extensive cell death in intact and injured knees, and that early treatment interventions should be aimed at preventing chondrocyte death.

Figure 1

(a) Exposed mouse knee preparation showing the medial tibial plateau (T), and the medial femoral condyle (F) with the meniscus removed. Scale bar = 1000 µm. (b) 3D image of the cartilage showing the injury (black middle line, ~20 µm width, ~350 µm length) and the zonal sections used to count the cell death. Scale bar = 100 µm and the spacing between yellow lines = 100 µm.

Figure 2

Flow charts illustrating the experimental groups.

Figure 3

(a) Mean percentage of dead chondrocytes as a function of time in the six different groups; 2 non-loaded control groups LMI, LMI and 4 loaded groups LMI, LMI, LMI, LMI), Bars = SD. (b) Significant difference in cell death, green (P < 0.001), black (P ≤ 0.05).

Figure 4

3D image of the cartilage showing the cartilage injury (~20 µm width, ~350 µm length), live (green), dead (red) cells and collagen tissue (grey) for three different times. The intact menisci prevent cell death in LMI group (a–c) while cell death increased rapidly for the corresponding conditions with the meniscectomy in LMI animal group (d–f). Bar = 80 µm.

Figure 5

3D image of the cartilage showing live cells (green), dead cells (red), and collagen tissue (grey) for three time points in the protocol. The intact menisci prevent cell death in the LMI group (a–c) while cell death increased uniformly across the cartilage surface for the corresponding conditions in the LMI animal group (d–f). At 240 min, cell death approximately doubled in the LMI joints compared to the LMI joints. Bar = 60 µm.

Figure 6

Mean percentage of dead chondrocytes as a function of distance from the cartilage injury (middle line) for three different groups (a) LMI; n = 8, (b) LMI; n = 8 and (c) LMI; n = 8 at three different times. (d) Percentage of cell death for the three groups LMI, LMI and LMI at 240 min. Bars = SD.

Figure 7

Release of cartilage fragments appeared clearly on the anterior side of the cartilage injury at 240 min or 120 contractions when the medial meniscus was removed (LMI animal group).

Figure 8

3D image of the cartilage showing necrotic (green) and apoptotic (red) cells at two time points (a) 120 min and (b) 240 min. Both images were taken from the LMI animal group. A single apoptotic cell was seen at 120 min (white arrow) near the cartilage injury (yellow arrow). Apoptotic cells start to progress from near the cartilage injury towards regions away from the injury at 240 min.