New morphological evidence of the ‘fate’ of growth plate hypertrophic chondrocytes in the general context of endochondral ossification

The ‘fate’ of growth plate hypertrophic chondrocytes has been long debated with two opposing theories: cell apoptosis or survival with transformation into osteogenic cells. This study was carried out on the proximal tibial growth plate of rabbits using light microscopy, scanning and transmission electron microscopy. We focused particularly on the orientation of the specimens included in order to define the mineral deposition and the vascular invasion lines and obtain histological and ultrastructural images at the corresponding height of the plate. Chondrocyte morphology transformation through the maturation process (characterized by vesicles and then large cytoplasmic lacunae before condensation, fragmentation and disappearance of the nuclear chromatin) did not correspond to that observed in the ‘in vitro’ apoptosis models. These findings rather suggested the passage of free water from the cartilage matrix into a still live cell (swelling). The level of these changes suggested a close relationship with the mineral deposition line. Furthermore, the study provided evidence that the metaphyseal capillaries could advance inside the columns of stacked hypertrophic chondrocytes (delimited by the intercolumnar septa) without the need for calcified matrix resorption because the thin transverse septa between the stacked chondrocyte (below the mineral deposition line) were not calcified. The zonal distribution of cell types (hypertrophic chondrocytes, osteoblasts, osteoclasts and macrophages) did not reveal osteoclasts or chondroclasts at this level. Morphological and morphometric analysis recorded globular masses of an amorphous, necrotic material in a zone 0–70 μm below the vascular invasion line occasionally surrounded by a membrane (indicated as ‘hypertrophic chondrocyte ghosts’). These masses and the same material not bound by a membrane were surrounded by a large number of macrophages and other blood cell precursors, suggesting this could be the cause of macrophage recall and activation. The most recent hypotheses based on genetic and lineage tracing studies stating that hypertrophic chondrocytes can survive and transform into osteoblasts and osteocytes (trans-differentiation) were not confirmed by the ultrastructural morphology or by the zonal comparative counting and distribution of cell types below the vascular invasion line.