The Journal of Bone and Joint Surgery, Vol 67, Issue 3 455-464, Copyright © 1985 by Journal of Bone and Joint Surgery, Inc
Ultrastructural modifications of proteoglycans coincident with mineralization in local regions of rat growth plate
N Shepard and N Mitchell
We carried out histochemical and electron-microscopic studies to examine
the relationship between mineral deposition and changes in the organization
of proteoglycans during mineralization in the cartilaginous growth plate of
the rat. To preserve the distribution and organization of proteoglycans in
the extracellular matrix and to stain proteoglycans, acridine orange was
included in the solutions that were used for fixation and demineralization.
In undecalcified sections, mineral crystals that initially appeared at the
level of the penultimate hypertrophic chondrocyte grew into dense,
spherical mineral clusters, roughly one micrometer in diameter, in the
longitudinal septa of the lowermost hypertrophic zone. Following the
removal of mineral with EDTA in the presence of acridine orange,
proteoglycan structures with a characteristic rosette-like architecture
were revealed within the matrix of the longitudinal septa of the lower
hypertrophic zone. The location of these rosette-like proteoglycan
structures was identical to that of the dense, spherical mineral clusters
in the undemineralized controls, and they were remarkably similar in
morphology and size. In the electron micrographs stained with uranyl
acetate and lead citrate, the greatly increased density of the rosette-like
structures and large diameter of the proteoglycan structures located in
mineral clusters contrasted sharply with the slender strands of more
faintly stained proteoglycan that was diffusely distributed throughout most
of the extracellular matrix. X-ray microprobe analysis for sulphur
confirmed the existence of proteoglycans in the rosette-like structures and
demonstrated that the concentration of proteoglycans was selectively
increased in these regions. The cores of the metaphyseal calcified
cartilage also exhibited rosette-like proteoglycan structures, which before
demineralization were totally obscured by the diffuse, dense homogeneous
deposition of mineral. Without stabilization of the proteoglycans in the
extracellular matrix with acridine orange, the rosette-like proteoglycan
structures could not be demonstrated. These results clearly indicate that
there is a selective increase in the concentration of proteoglycans in
exactly the same regions where mineral clusters are formed in the
hypertrophic zone of the cartilaginous growth plate.
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