Age and pattern of the onset of differential growth among growth plates in rats

Abstract

Differential growth is the phenomenon whereby growth plates in the same individual at the same time all have uniquely different axial growth velocities. Differential growth is clearly present in the adolescent skeleton. In this study we ask two questions. When and by what pattern does the phenomenon of differential growth begin? Second, to what extent are the development of differential growth velocities correlated with changes in hypertrophic chondrocyte volume and/or with changes in chondrocytic production/turnover? Four growth plates (proximal and distal radial; proximal and distal tibial) were studied at 24 different time points in Long-Evans rats between the 17th gestational day (when differential growth does not exist) and postnatal day 27 (when differential growth is well established). Growth velocities were measured using fluorochrome labeling. Using stereological methodology, multiple chondrocytic kinetic parameters were measured for all growth plates. Elongation of the proximal radial growth plate decreases relative to elongation in the other three growth plates in the late fetal phase. Differential growth is fully expressed at postnatal day 13 when the other three growth plates start to decrease daily elongation at different rates. Differential growth is primarily associated with differences in hypertrophic cell volume manifested when growth deceleration occurs. This study also illustrates that differential growth is superimposed on systemic regulators that affect all growth plates simultaneously. The most dramatic illustration of this is the sharp decline in growth velocity in all four growth plates that occurs perinatally.

Figure 1

Micrograph of the distal radial growth plate viewed under epifluorescence microscopy (250μm scale bar included). This micrograph illustrates the technique of fluorochrome labeling to measure growth velocity. At the time of injection the fluorochrome label is permanently incorporated and fixed in a narrow band at the mineralizing front at the chondro-osseous junction. With time (in this example 24 hours) and continued new endochondral ossification, the chondro-osseous junction separates from the fluorochrome label by a distance equal to the growth velocity divided by time interval. The half-life of calcein in the blood is estimated to be 7 minutes . When measuring the distance to the chondro-osseous junction we used the leading edge (edge furthest from the growth plate) of the fluorochrome label as a starting point. This basic technique can be modified to include double labels with different fluorochromes such as oxytetracycline, alizarine complexone or calcein using correspondingly appropriate epi-fluorescent filters. Time intervals must be a multiple of 24 hours to cancel out any circadian effect. Other metaphyseal fluorescence represents autoflourescence.

Figure 2. Rates of Growth in Four Different Growth Plates in Fetal and Neonatal Rats (μm / 24 hours)

Pattern of changes in growth velocity in four different growth plates. On the 17^th –18^th day of gestation (day −4) all growth plates are elongating at ~200μm/da. In the late fetal period the proximal radial growth plate velocity is significantly slower than the other 3 growth plates. By postnatal day 13 all four growth plates have significantly different growth velocities. Significant deceleration in birth growth velocities occurs in all four growth plates perinatally. Bars = ± standard error.

Figure 3. Volumes of Hypertrophic Chondrocytes in Four Different Growth Plates in Fetal and Neonatal Rats (μm³)

Changes in hypertrophic chondrocyte volumes in four different growth plates over the course of this study. The changing pattern in cellular volumes in the postnatal adolescent period parallels changes in growth velocity during the same period.

Figure 4. Chondrocytes Produced / Turned Over in Four Different Growth Plates in Fetal and Neonatal Rats (chondrocytes / 24 hours)

Changes in chondrocyte production / turn over in four different growth plates over the course of this study. The changing pattern in the prenatal period parallels changes in growth velocity during the same period.

Figure 5

The three micrographs are of 1.5um thick plastic-embedded sections stained with methylene blue/azure II/ basic fuchsin of the distal radial growth plate (scale bar = 200um). Three time points for illustration where chosen by referring to Figure 2 and drawing an imaginary line parallel with the x-axis at about ~350um/day, and noting time points where the growth velocity for the distal radial growth plate crossed this line. Three of these time points (day 20 gestation, and days 13 and 18 postnatal) are illustrated in Figure 5. The significant point is that all three micrographs illustrate the same growth plate elongating at ~350um/day but at three different time points in its biological life span. What differs at each time point is the primary variable by which this growth velocity is achieved. At the earliest time point (left, day 20 gestational), mean hypertrophic cell volume is low (9,540μm³); however, the number of chondrocytes being produced is high (19,070 cells/day). In contrast, at the latest illustrated time point (right, day 18 postnatal), mean hypertrophic cell volume is high (14,640μm³) while the number of cells turned over is only 12,710 cells per day. The micrograph in the center at 13 days of age represents the chronological point of emergence of differential growth. Visual comparison on the micrographs on the right and left illustrate an increase in matrix volume per hypertrophic chondrocyte (3,520μm³ versus 6,240μm³) , , . Raw data and calculations are not included in this manuscript. This is consistent with what is expected if “matrix directed cellular swelling” , is the chief engine of growth . This hypothesis requires an adequate volume of biomechanically normal matrix to direct the chondrocytic shape change that accompanies the increase in cellular volume of chondrocytes as they hypertrophy .