Matrix resorption in endochondral bone growth : ultrastructural studies, with special attention to the chondroclast

Abstract

A substantial amount of the calcified cartilage in the distal parts of the epiphyseal plate is resorbed during endochondral growth of long bones. However, the mechanisms involved in this process are still largely unknown. On the other hand, bone-resorption in the lower metaphysis has been thoroughly investigated. Since bone and cartilage are tissues with different composition, our hypothesis was that the resorption mechanisms may also be separate. At the epiphyseal/metaphyseal border, cells with a morphology resembling the osteoclasts are present. They are by some authors designated chondroclasts. However, their specific properties regarding morphological and functional features have not received much attention.

In the present study, Sprague-Dawley rats were used to investigate some aspects of matrix resorption in calcified cartilage compared with bone. Four groups of animals were analyzed mainly at the ultrastructural level; one normal group, one group with impaired tissue resorption due to nutritionally induced vitamin D deficiency rickets, and two groups of animals healing from rickets at 48 or 72 hours after introduction of normal food. Our results are based on a number of measurements of cellular parameters at the ultrastructural level and expression of markers for chondroclasts/ostcoclasts; ie tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We also wanted to clarify the role of the extracellular matrix proteins bone sialoprotein, osteopontin and fibronectin. Since they all contain an Arg-Gly-Asp (RGD) sequence, they may bind to integrin receptors on cell surfaces, making them potentially active in cell-matrix adhesion, as an initial step in the resorption sequence.

Our approach with quantitative morphology and immunohistochemistry was aimed at demonstrating even smaller differences between cells and tissue compartments than are normally detected by qualitative means. In summary we showed that chondroclasts and osteoclasts were different regarding some characteristic morphometric features, indicating differences in resorption mechanisms between bone and cartilage. Furthermore, we identified a previously unrecognized structure at the cell-matrix border with features of both ruffled borders and clear zones, i.e. the traditional resorption characteristics. This structure was designated the intermediate zone, suggested to be a transient stage in the development of ruffled border within an established clear zone.

Bone sialoprotein located outside the clear zone of chondroclasts has a potential role in interaction between these cells and their adjacent matrix. Fibronectin is associated with collagen type I in bone and may be involved in collagen fibril formation or mineralization, but is apparently not involved in cell-matrix interaction in highly differentiated cartilage or bone. The role of osteopontin, observed in the ruffled border area of chondroclasts, remains to be clarified. In rickets, clast-like cells are able to establish a cell-matrix interaction. However, development of the ruffled border is interrupted, paralleled by absence of extracellular enzyme release onto the matrix surface.

Restitution of traditional ultrastructural resorption characteristics, at 48 hours after introduction of normal food, coincides largely with secretion of cathepsin K to the matrix surface, rendering this enzyme suitable as a marker for active resorption. As TRAP was not secreted in any significant amounts until at 72 hours, this enzyme may be involved in termination of the resorption sequence.