Hypertrophic Zone. The functions of this zone are preparation of the matrix for expansion, degradation, and matrix calcification. Chondrocytes in this zone progressively become more spherical and greatly enlarged along their vertical axis. By the bottom of the zone, they have enlarged to five times their size in the proliferative zone. The cytoplasm of chondrocytes in the top half of the zone stains positively for glycogen; near the middle of the zone it abruptly loses all glycogen-staining ability.

On electron microscopy, chondrocytes in the top half of the hypertrophic zone appear normal and contain the full complement of cytoplasmic components, but in the bottom half of the zone the cytoplasm contains numerous vacuoles that can occupy over 85% of the total cytoplasmic volume.

The last cell at the base of each cell column is clearly nonviable and shows extensive fragmentation of the cell membrane and the nuclear envelope, with loss of all cytoplasmic components except a few mitochondria and scattered remnants of endoplasmic reticulum. Mitochondria and cell membranes of chondrocytes in the top half of the hypertrophic zone are loaded with calcium. Toward the middle of the zone, mitochondria rapidly lose calcium, and at the bottom of the zone, both mitochondria and cell membranes have no calcium. These findings suggest that mitochondrial calcium may be involved in cartilage calcification.

The hypertrophic zone is avascular, and PO₂ is therefore low. In the bottom half of the zone, glycogen is completely depleted. There is no other source of nutrition to serve as an energy source for the mitochondria. Because calcium uptake and retention require energy, as soon as the chondrocytes’ glycogen supplies are exhausted, mitochondria release calcium, a factor that may play a role in matrix calcification.

The metabolic events in the proliferative and hypertrophic zones can be summarized as follows. In the proliferative zone, PO₂ is high, aerobic metabolism occurs, glycogen is stored, and mitochondria form ATP. In the hypertrophic zone, PO₂ is low, anaerobic metabolism occurs, and glycogen is consumed until near the middle of the zone, where mitochondria switch from forming ATP to accumulating calcium. ATP formation and calcium accumulation cannot take place simultaneously. Both processes require energy, which comes from the respiratory chain in the mitochondria. In addition, ATP formation requires the presence of ADP, whereas calcium accumulation does not. Possibly, in the hypertrophic zone, there is insufficient ADP for significant ATP formation.

The matrix of the hypertrophic zone shows positive histochemical reactions for hyaluronan and proteoglycan degradation products (see Plate 2-25). From the reserve zone through the hypertrophic zone there is a progressive decrease in the length of proteoglycan aggregates and in the number of aggregate subunits in the matrix. The distance between the subunits also increases.

The large proteoglycan aggregates with tightly packed subunits suppress mineralization and its spread, whereas smaller aggregates with widely spaced subunits at the bottom of the hypertrophic zone tend to be less effective in preventing mineral growth. In any event, proteoglycan disaggregation and degradation must take place before significant mineralization occurs.

The initial calcification (seeding, or nucleation) in the bottom of the hypertrophic zone, called the zone of provisional calcification, occurs within or on vesicles in the longitudinal septa of the matrix (see Plate 2-16). Matrix vesicles are densest in the hypertrophic zone. They are small, trilamellar membrane structures (100 to 150 nanometers in diameter) released by chondrocytes. Matrix vesicles are rich in various phosphatases, some of which act as a pyrophosphatase to destroy pyrophosphate, another inhibitor of calcium-phosphate precipitation. Matrix vesicles begin to accumulate calcium at the same level in the hypertrophic zone at which mitochondria begin to lose it. This suggests that mitochondrial calcium is involved in the initial calcification in the growth plate. Initial calcification, whether it is within or on matrix vesicles or collagen fibers, may be in the form of amorphous calcium phosphate, but this mineral phase rapidly transitions to hydroxyapatite crystal formation. With continued crystal growth and confluence, the longitudinal septa become gradually calcified.