Differentiation and growth of chondrocytes in fetal growth plates of vertebrate 
long bones and ribs appear to occur in a gradual, continuous manner between the 
resting zone through the proliferation zone, maturation zone, and upper and 
lower hypertrophic zones, with a continuous increase in cell size up to 10-fold 
of the volume of a resting chondrocyte. Here we provide evidence, however, that 
after centrifugation through a continuous Percoll gradient growth plate 
chondrocytes separate into four distinct cell populations (B1 to B4) which 
differ markedly in density, size, and gene expression. These populations collect 
in the absence of any phase borders in the gradient which might serve as 
concentration barriers. Fractions B1 and B2 contained the largest cells with the 
lowest buoyant density and showed the highest expression levels for type X 
collagen (Col X), but only the B1 population expressed high levels of matrix 
metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly 
smaller and expressed little Col X, while cells in fraction B4 were of similar 
size to cells in the resting zone without significant Col X expression. The 
highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor 
(PTHR-1), and Indian hedgehog (Ihh) expression were also found in the 
hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as 
expected from in situ hybridization data on PTHR-1 expression in fetal rodent or 
chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal 
fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and 
PTHR-1 by more than 50% and the expression of Ihh nearly completely. In 
contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently 
stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These 
findings confirm the existence of distinct differentiation stages within 
chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp 
et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP 
fragments controlling the differentiation of proliferating to prehypertrophic 
chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic 
chondrocytes.