Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates



Regulation of postnatal growth of long bones occurs in multiple levels of chondrocytic activity, including stem cell proliferation, proliferative zone cycling, and regulation of changes in chondrocytic shape during hypertrophy. The differentiation sequence of chondrocytes is the same in all growth plates, but rates of elongation at a single point in time and over a period of time differ widely among individual growth plates, which suggests that the rates of sequential gene activation and suppression in this phenotypic pattern can vary. The purpose of this study was to investigate, directly and in vivo, parameters of the cell cycle of proliferative chondrocytes in growth plates growing at widely different rates at a single point in time in order to analyze the relationship between cell cycle time, including the duration of each phase of the cell cycle (G1, S, G2, and M), and the rate of growth. The experimental design used repeated pulse labeling with bromodeoxyuridine and was analyzed using a regression model of time of pulse label with increasing labeling index. Total cell cycle time was calculated as the inverse of the slope of the relationship of the labeling index and the time between labels. The y intercept was the calculated labeling index at time zero. Multiple comparison contrasts were used to test for individual differences among four growth plates with growth rates ranging from approximately 50 to 400 μm per 24 hours from 28-day-old rats. The estimate of total cell cycle time for the proximal tibial growth plate was 30.9 hours. Cell cycle times for the other three growth plates were 34.0, 48.7, and 76.3 hours for the distal radius, distal tibia and proximal radius, respectively. Although the times for the proximal tibia and distal radius did not differ significantly, all other times were significantly different (p < 0.05) Almost all differences in total cell cycle time were attributable to significant differences in the length of the G1 phase. The S phase was estimated at 3.4–6.1 hours; the G2 phase, at 3.0 hours; and the M phase, at 0.5-0.6 hours. The current study suggests that regulation through cell cycle parameters, specifically in the G1 phase, may be involved in overall regulation of differential postnatal long bone growth. It has previously been established that increase and shape change of cellular volume during hypertrophy may be regulated at the level of individual growth plates and that both are significant in understanding differential growth of long bone at this level. By demonstrating that chondrocytes in the proliferating zone have different cell cycle times that are regulated primarily through differences in the duration of G1, this study suggests that, in addition to systemic controls of chondrocyte proliferation, local controls may modulate rates of proliferation of individual growth plates and thus may be another locally mediated regulator of differential growth.