Morphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage

Abstract

Recent biochemical and morphologic evidence supports the concept that hypertrophic chondrocytes of growth plate cartilage are fully viable cells that play a major functional role in controlling endochondral ossification. However, events associated with chondrocyte death remain unknown. In this study we assess the viability of terminal hypertrophic chondrocytes in situ in an organ culture system viewed simultaneously with rectified Nomarski interference contrast optics and with vital staining under fluorescence optics. Second, we use two methods of optimal chemical fixation at the ultrastructural level to define morphologically distinct stages of the terminal hypertrophic chondrocyte, which we interpret as the stages preceding chondrocyte death. An analysis of serial sections at the light microscope level showed that terminal chondrocytes were found, with different probabilities, in three morphologically distinguishable stages. Seventy-five percent of all profiles were fully hydrated cells with an intact plasma membrane making direct contact with the pericellular matrix, a morphology identical to that of living terminal chondrocytes viewed in Nomarski optics. Approximately 1% of terminal chondrocytes, while still in a fully hydrated state, consistently made a direct asymmetrical contact of the plasma membrane with the last transverse septum. In 24% of the profiles, terminal chondrocytes were found as condensed cells that retained their attachment to the last transverse septum. The stages were not characteristic of chondrocytes positioned more proximally in the growth plate. We hypothesize that a condensed morphology eventually characterizes each hypertrophic chondrocyte, and we relate these observations to current hypotheses concerning the mechanism of death of hypertrophic chondrocytes.

Chondrocyte apoptosis in endochondral ossification of chick sterna

“…In Figure 2B most stages of this process are visible in adjacent chondrocytes at the interface of cartilage and invading vascular cells. Minor retraction of the chondrocytes from the extracellular matrix, as previously seen in pig growth plate (Farnum and Wilsman, 1987), was suggestive of a n early stage of chondrocyte apoptosis. Matrix in this region appears less dense and the space generated by contraction of the chondrocyte is filled with a loose electron dense fibrous mesh.…”

“…Electron microscopy of the primary ossification center of the chick sterna showed a preponderance of hypertrophic chondrocytes indistinguishable in appearance from those previously described in growth plate cartilage (Farnum and Wilsman, 1987) (Fig. 2A).…”

“…More extensive chondrocyte condensation involved extensive vacuole formation and development of a dense crenated nucleus. As described by Farnum and Wilsman (1987) in growth plate the condensed chondrocytes often appeared to be more closely attached to the extracellular matrix separating it from invading vascular cells. Formation of the final morphological stage of apoptosis, a cluster of vesicles (apoptotic bodies), was apparent as cellular debris, mostly vesicular in nature, in otherwise empty chondrocyte lacunae.…”

“…The proportion of chondrocytes with a condensed morphology was determined by counting condensed and normal chondrocyte profiles in sections taken every 100 pm through the primary ossification center. Using this procedure the proportion of chondrocytes with a condensed profile was underestimated, since the probability of finding the smaller condensed chondrocyte profile is less than that of finding the larger hydrated cell (Farnum and Wilsman, 1987).…”

“…Growth is produced by chondrocyte division, matrix production, and chondrocyte hypertrophy. Resorption of the growth plate at the interface with the metaphyseal vasculature is coordinated with tissue expansion and associated with rapid chondrocyte death adjacent to the invading vasculature (Farnum and Wilsman, 1987). Subsequently the transverse septa separating terminal chondrocytes from invading vasculature are resorbed, blood vessels invade the vacated cell lacunae, and progressive vascular invasion through columns of hypertrophic chondrocytes follows (Bettex-Galland et al, 1990;Hunziker, 1994).…”