Ultrastructural modifications of proteoglycans coincident with mineralization in local regions of rat growth plate.

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.