Session II - Basic Science

Chondrocyte Apoptosis after Articular Cartilage Injury

Joseph Borrelli, Jr., MD; William Ricci, MD; Meghan Burns, BS; Paul Swanson, MD; Richard Hotchkiss, MD, Washington University School of Medicine, St. Louis, MO (supported by OTA Research Grant)

Purpose: Apoptosis or "programmed cell death" is known to play a role in tissue development, aging, and disease. Apoptosis occurs via highly regulated pathways that involve an initiating stimulus, intracellular signals, and the expression of well-defined genes. Chondrocytes undergoing apoptosis have been found in osteoarthritic cartilage and after acute articular cartilage injury in in-vitro experiments. These experiments, using chick sternal cartilage and bovine articular cartilage, identified chondrocyte apoptosis after trephine wounding and after blunt impact. This study was undertaken to determine whether chondrocytes could be stimulated to undergo apoptosis as a result of blunt-impact load in an in-vivo rabbit model.

Methods: Three-month old, New Zealand White rabbits (3 kg) underwent a posterior surgical approach to the posterior aspect of the medial femoral condyle. The articular surface was impacted with use of a previously validated pendulum type device and aluminum impactor. The contralateral limb served as the control in which a sham operation, including an arthrotomy, was performed. Tissue sections were examined 7 to 10 days after injury. Four methods, differing in sensitivity and specificity, were employed to detect apoptosis in each tissue section: conventional light microscopy, terminal deoxynucleotide transferase mediated dUTP nick end labeling (TUNEL), transmission electron microscopy (TEM), and immunohistological analysis. Specimens were evaluated by an observer familiar with the microscopic appearance of apoptosis, but blinded to the specimen identity. For TUNEL and immunohistologically stained specimens, a minimum of five fields were evaluated randomly at 200¥ magnification using a Nikon RCM 8000 laser-scanning confocal microscope. Specimens for TEM evaluation were stained with uranyl acetate and lead citrate; ultrathin (60 nm) sections were mounted and examined with a Philips CM 10 transmission electron microscope.

Results: Cartilage explants from the impacted area of each animal showed increased numbers of chondrocytes undergoing apoptosis in each of the cartilage zones when compared to the cartilage from the sham operations. Specimens evaluated with H&E staining and light microscopy demonstrated apoptotic chondrocytes with condensed and pyknotic nuclei and alterations in the typical staining characteristics of normal cytoplasm. The cytoplasm was typically found to be disorganized and contained large lipid vacuoles. In many cases the cytoplasm was typically shrunken in volume and retracted from the pericellular matrix. These cells were more commonly seen in the middle and deep cartilage zones, but were also observed in the superficial and calcified cartilage regions as well. Immunohistological analysis of the specimens revealed numerous chondrocytes, particularly in the middle and deep zones, with condensed chromatin and a few chondrocytes with fragmented nuclei. The presence of DNA breaks was confirmed by TUNEL in many chondrocytes within the impacted cartilage as compared to the cartilage from the sham operation. The presence of morphologically altered chondrocytes in the impacted cartilage specimens was confirmed by TEM. Although bona fide apoptotic bodies with accompanying external membrane blebbing were not observed, the apoptotic cells did have condensed chromatin, disorganized cytoplasm, and large intra-cytoplasmic lipid collections and volume shrinkage.

Discussion: The results of this study show that: 1) chondrocyte apoptosis can be induced by blunt impact load on intact articular cartilage; 2) this apoptotic response is localized primarily to the middle and deep zones of the cartilage; and 3) in-vivo chondrocyte apoptosis, resulting from mechanical injury, exhibits cellular changes similar to those demonstrated in in-vitro studies. On the basis of these findings, it can be hypothesized that apoptosis may play a role in cartilage degradation after articular cartilage injury, although its exact contribution is unknown.