PROGRESSION AND RECAPITULATION OF THE CHONDROCYTE DIFFERENTIATION PROGRAM - CARTILAGE MATRIX PROTEIN IS A MARKER FOR CARTILAGE MATURATION

During endochondral bone formation, chondrocytes in the cartilaginous anlage of long bones progress through a spatially and temporally regul ated differentiation program before being replaced by bone. To underst and this process, we have characterized the differentiation program an d analyzed the relationship between chondrocytes and their extracellul ar environment in the regulation of the program. Our results indicate that, within an epiphyseal growth plate, the zone of proliferating cho ndrocytes is not contiguous with the zone of hypertrophic chondrocytes identified by the transcription of the type X collagen gene. We find that the postproliferative chondrocytes which make up the zone between the zones of proliferation and hypertrophy specifically transcribe th e gene for cartilage matrix protein (CMP). This zone has been termed t he zone of maturation. The identification of this unique population of chondrocytes demonstrates that the chondrocyte differentiation progra m consists of at least three stages. CMP translation products are pres ent in the matrix surrounding the nonproliferative chondrocytes of bot h the zones of maturation and hypertrophy. Thus, CMP is a marker for p ostmitotic chondrocytes. As a result of the changes in gene expression during the differentiation program, chondrocytes in each zone reside in an extracellular matrix with a unique macromolecular composition. C hondrocytes in primary cell culture can proceed through the same diffe rentiation program as they do in the cartilaginous rudiments. In cultu re, a wave of differentiation begins in the center of a colony and spr eads to its periphery. The cessation of proliferation coincides with t he appearance of CMP and eventually the cells undergo hypertrophy and synthesize type X collagen. These results reveal distinct switches at the proliferative-maturation transition and at the maturation-hypertro phy transition during chondrocyte differentiation and indicate that ch ondrocytes synthesize new matrix molecules and thus modify their preex isting microenvironment as differentiation progresses. However, when ' 'terminally'' differentiated hypertrophic chondrocytes are released fr om their surrounding environment and incubated in pellet culture, they stop type X collagen synthesis, resume proliferation, and reinitiate aggrecan synthesis. Eventually they cease proliferation and reinitiate CMP synthesis and finally type X collagen. Thus they are capable of r ecapitulating all three stages of the differentiation program in vitro . The data suggest a high degree of plasticity in the chondrocyte diff erentiation program and demonstrate that the progression and maintenan ce of this program is regulated, at least in part, by the extracellula r environment which surrounds a differentiating chondrocyte during end ochondral bone formation. (C) 1995 Academic Press, Inc.