Stereological and serial section analysis of chondrocytic enlargement in the proximal tibial growth plate of the rat.

BACKGROUND: It has been suggested that within the growth plate, the final volume and shape of hypertrophic chondrocytes are important variables in determining the rate of longitudinal bone growth. To better understand the organization and regulation of chondrocytic hypertrophy as related to longitudinal bone growth, the beginning and end, and the location and magnitude of chondrocytic volume and shape changes during the hypertrophic process were defined in the proximal tibial growth plate of 35-day-old rats. METHODS: In this study we used two different approaches, a stereological analysis of chondrocytes in unbiasedly defined, narrow growth plate strata, and a serial section reconstruction and measurement of individual cells. In both experiments chondrocytes were preserved using optimal chemical fixation. Proliferating chondrocytes were identified using bromodeoxyuridine labelling, and the rate of longitudinal bone growth was determined using oxytetracycline labelling. RESULTS: In both studies, immediately following cell division in the proliferative zone, chondrocytic volume gradually increased toward the mid-point of the growth plate. During this phase of about 30 hours, approximately 20% of the final cell volume was obtained. During the following 20 hours the remaining 80% was acquired. The estimated rate of cell volume increased changed from approximately 50 microns 3/hr during the first 30 hours to about 800 microns 3/hr during the last 20 hours. The increase in cell volume resulted in an increase in both the vertical and the horizontal chondrocytic diameters. Cell parameters did not change during the final five hours of the maturation process. CONCLUSIONS: In this study we demonstrated that chondrocytic enlargement starts immediately following cell division in the proliferative zone, and that chondrocytic enlargement consists of two morphologically distinguishable phases. The transition point between the first and the second phase of chondrocytic enlargement corresponded with the junction between the proliferative zone and the maturation zone.

Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography.

The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.