CeN Tissue Kinet. (1974) 7, 285-291.

COMPENSATORY GROWTH I N THE RAT TIBIA

A N N E DAWSON A N D N. F. KEMBER

The Medical College of Saint Bartholomew's Hospital, London

(Received 25 October 1913; revision received I December 1973)

ABSTRACT

When the proximal cartilage plate of one tibia of a rat is sterilized by radiation, compensatory growth occurs at the distal plate of the same bone. This growth is marked by small changes in the cell kinetic parameters and histology of the growth cartilage when compared with the distal cartilage plate in the unirradiated tibia. The changes are consistent with a delay in the maturation of the plate showing compensatory growth. Some possible mechanisms are considered, but the evidence available at present does not give decisive support to any particular theory.

I N T R O D U C T I O N

Reidy et al. (1947) found compensatory growth in the distal epiphysis of the tibia of a dog when the growth of the proximal epiphysis had been sterilized by radiation. Their work was confirmed in rabbits by Hall-Craggs & Lawrence (1969) who found that a similar effect was obtained when the proximal epiphyseal plate was fixed by stapling. They also reported that no compensatory growth was found in the distal plate of the femur indicating some control mechanism internal to one bone that does not affect growth in adjacent bones.

We wished to study these changes at the cellular level, and have undertaken two studies: the first a pilot radiographic experiment designed to confirm the existence of the effect in the rat, and the second an examination of the cell kinetics and histology of the compensatory system, during the period from the irradiation of the proximal plate to the closure of the distal plate.

MATERIAL A N D METHODS

In all experiments, male Wistar rats, 4 weeks old and weighing 50-60 g, were used. A group of six rats was taken for a radiographic study of bone growth. A small stainless steel pin was placed in the lower third of each tibia of these animals. One week after operation, the right knee joint was irradiated locally with 2400 rads of 250 kV X-rays (150 rads/min, HVT 1.2 mm Cu) through a 2 x 2.5 cm aperture in a lead sheet placed directly above the knee joint. This dose was chosen since our experience with cell survival studies indicated that it would be sufficient to sterilize the proximal growth plate (Kember, 1967). After the irradiation

Correspondence : Dr N. F. Kember, The Medical College of Saint Bartholomew's Hospital, Charterhouse Square, London, E.C.I.

285

286 Anne Da~.son and N . F. Kember the animals were radiographed weekly for 10 weeks so that measurements of bone growth could be made. The animals were positioned under light ether anaesthesia with the tibiae parallel to the film at 80 cm focus distance. The dose per radiograph was less than 0.2 rads. Three measurements were made on the radiographs: the overall length of the tibia, the distance from the pin to the proximal end, and the distance from the pin to the distal end of the bone. The measurements were made with dividers and a steel rule.

Secondly, four groups of four rats were given the same radiation dose to the right knee joint preparatory to autoradiographic studies. Four animals were injected intraperitoneally at each of the time intervals 1, 4, 6 and 8 weeks after irradiation, with tritiated thymidine (0.5 pCi per g body weight, specific activity 5 Ci/mole, from the Radiochemical Centre, Amer- sham). All the rats were killed at 1 hr after the 3H-TdR injection, both tibiae were excised, decalcified in EDTA and blocked in paraffin wax. Five micron sagittal sections were cut and coated with Ilford K5 emulsion. The autoradiographs were exposed for 5 weeks and stained with haematoxylin and eosin after development.

RESULTS

One rat in the radiographic group became sick and lost weight so that it was excluded from the series. One rat suffered a spontaneous fracture of the femur at 6 weeks after irradiation and two more at 2 months after irradiation. These animals were killed.

The contributions to bone growth of the proximal and distal plates of the irradiated and non-irradiated tibiae, as measured radiographically, are shown in Fig. 1. The data are plotted as increase in length relative to the pin for time intervals after irradiation, with differential growth rate curves for the distal plates below. These measurements showed that growth at the proximal plate of the irradiated tibia ceased after about 10 days and that the plate did not recover. The autoradiographs of the irradiated proximal plates showed a lack of columnar structure, a few abnormal giant cells, and the complete absence of cells labelled with 3H-TdR. Compensatory growth of the distal plate on the irradiated side is evident from Fig. 1, and all differences between the left and right distal length curves were found to be significant at the 5 % level for the intervals of 4 weeks or more after irradiation.

Since the rats were killed at 1 hr after the 3H-TdR injection, most of the cells seen on the autoradiographs were still in DNA synthesis phase. The autoradiographs of the distal epiphyses were scanned, and the percentages of labelled cells in the proliferation zones of the cartilage plates were calculated. At least loo0 nuclei were observed, the criterion for labelling being five grains per nucleus against a background of one to two grains per nucleus, although most of the cells counted were heavily labelled. The positions of 100 labelled nuclei were also recorded in order that labelling profiles could be constructed. The labelling profile is a histo- gram showing the frequency of occurrence of labelled cells at the various positions down the cell columns, starting with the first cell at the epiphyseal side of the cartilage plate. The effective size of the proliferation zone can be calculated from the labelling profile (Kember, 1972), and the percentage of labelled cells within this zone gives the labelling index. The heights of twenty hypertrophic cells at the metaphyseal border of the growth plate were measured using a calibrated eye-piece graticule so that the mean contribution of each cell to overall bone growth could be calculated.

The labelling profiles and indices presented in Fig. 2 combine the data from the four rats in each group. At 1 week after irradiation, labelling profiles from the distal plates of the

Compensatory growth in the rat tibia ' 1 Proximal end

of tibia

10

t

O E Distal end

287

Days after irradiation of right proximal plate

FIG. 1 . Upper graph: Growth contributions from right and left proximal and distal plates of rat tibia at intervals following irradiation of right proximal plate. Means for five animals with standard errors. Average length tibia at irradiation: 2.9 cm. Lower graph: Growth rate curves derived from the growth increase curves above.

irradiation, labelling profiles from the distal plates of the irradiated and unirradiated bones were similar, and the differences in the labelling indices were small. At 4 weeks aftcr irradiation there was still no detectable difference between the proliferation lengths of the right and left plates, but the labelling index on the irradiated side was 50 % higher. At 6 weeks after irradia- tion there was a clear difference in both the labelling indices and labelling profiles of the two plates. By 2 months after irradiation there were obvious differences in the histological appearances of the two plates (Figs. 3 and 4). No labelled cells were found in any of the plates on the left side, whereas two of the four rats killed at this interval showed 0.4% labelled cells in the persistent distal plates of the right side, although too few labelled cells were seen in these plates for a full labelljng profile to be drawn.

In Table 1 the cell kinetic data are given as mean values for the four rats killed at each time interval. The standard deviation is given for the labelling index, proliferation zone

288

Lef t

Atitie Dawsoti arid N . F. Keriiher

00

I 5 I0

Right Weeks after irrodiotion

I

5 2

I 5 10

8

Cell posillon down corliloge column

FIG. 2. Labelling profiles for distal plates showing positions of tritiatcd thyniidine labelled cells within cartilage columns: cell no. I adjacent to the epiphyseal border of the plate. Each histogram is drawn from the pooled results of four animals. The positions of 100 labelled cells in each plate were analysed. The profiles of the left and right plates are compared at each time interval after irradiation of the right proximal plate. The mean labelling index is given with each profile.

TABLE I . Cell kinetic data: distal cartilage plates; mean values for four aninials,except asshown, with standard deviations

Time after irradiation

Labelling Proliferation zone Hypertrophic Theoretical growth index (0,) length cells cell size (Am) rate* (,urn/day)

1 week Left

4 weeks Left

6 weeks Left

8 Iveeks Left

Right

Right

Right

Right

22 + 4 25 f 5 21 t 4 2 2 t 4 1 6 2 3 20 & 5 o + o

1 7 k 5

42.0 & 15.0 58.0 & 10.0 13.0 10.0 23.0 ? 16.0 0.2 * 0.02

13.0 +_ 9.0 0.0 & 0.0 0.5 & 0.5

* Calculation based on assumed DNA synthesis period of 6 hr duration (Kember, 1972). '1 Based on two rats only.

FIG. 3. Photomicrograph of right distal plate of tibia at 8 weeks after irradiation of the right proximal plate. The maturation is well advanced, but some cells are still arranged in short columns. Section stained with haematoxylin and eosin; magnification x 100.

FIG. 4. Comparative section from the distal end of left tibia from same rat as Fig. 3. The cartilage plate has been completely resorbed, and only the remains of cartilage are seen in the trabeculae.

290 Anne Daw5son and N . F. Kember length and hypertrophic cell size. A theoretical value for the growth rate of each plate cal- culated from the cell kinetic parameters (Kember, 1972) is given in this table. The differences in proliferation zone length and labelling index at 1 and 4 weeks after irradiation are small but they are cumulative in the calculation of growth rate. By 1 week after irradiation the growth of the right plate was 40% higher than that of the left, and at 4 weeks the calculated growth rate on the injured side was nearly double that on the control side. This finding agrees with the growth rates measured radiographically at 4 weeks. The greatest difference between growth rates of the right and left distal plates was found at 6 weeks after irradiation. In two out of the four rats killed at this interval there were no labelled cells in the plates of the left side, and the proliferation zone lengths in the left distal tibiae of the other two rats were about one third of the zone lengths in the right-hand bones.

By 8 weeks the distal plates on the control side had all closed (Fig. 4) and although there was evidence of growth in two of the four plates on the irradiated side, the closure of these plates was in progress (Fig. 3).

DISCUSSION

These results show that there is compensatory growth in the distal cartilage plate of the rat tibia when the proximal cartilage plate is damaged. Comparison of the growth curves (Fig. 1) reveals that this effect may appear as soon as 1 week after injury, but that it does not become significant until 4 weeks have elapsed. The compensatory effect is small, not more than a 50% increase in contribution to bone growth, and the changes in the cell proliferation patterns are correspondingly small (Table I) . However, the increased proliferative activity in the distal plate on the irradiated side is seen qualitatively in the comparative histology (Figs. 3 and 4) and confirmed quantitatively by the 3H-TdR labelling studies (Table 1).

The effect might be described as a delay in the maturation process of the distal plate, since during compensation the growth rate does not exceed its value at the time of irradiation, but shows a slower rate of decline than the growth rate on the unirradiated side. The kinetic data and the histological appearance are consistent with a lag in maturation. In this experi- ment the delay is about 2 weeks, but the magnitude of the effect may well depend on the age of the rat when the knee joint is irradiated.

It is possible that the insertion of metal markers into the cortex of the bones might have had a disturbing effect on nearby growth plates. However, it should have been the same in both tibiae. The rats showed a continuous increase in weight over the experimental period, apart from one whose weight fell between 2 and 4 weeks after irradiation, probably as a result of the accidental inclusion of a small section of the gut in the irradiation field. The irradiation also damaged the leg muscles, and the treated hind limb became contracted in comparison with the unirradiated limbs. However, the animals continued to walk freely, except in those animals where the femurs fractured.

The results agree in general with those of Reidy et al. (1947) on dogs, and of Hall-Craggs & Lawrence (1969) on rabbits. Since the bones of rats are smaller, the percentage errors in the differential measurements have been correspondingly larger but the results of our experiments are significant.

The results suggest a balance of growth between the two plates which is altered when one is damaged, and that therefore there is a degree of interdependence between the plates within one bone. Crilly (1972) has suggested that the two plates influence each other through the

Compensatory growth in the rat tibia 29 I periosteum. Since the effect has here been shown to result largely from delayed maturation of the growth plate, a slow acting control is suggested, and the observed changes could be effected through a growth inhibitor specific to growth cartilage in a manner analogous to that of the mitotic inhibitor in skin (Bullough, 1968). However, our results do not give conclusive support to either theory.

A C K N O W L E D G M E N T S

We would like to thank Professor H. A. B. Simons for his encouragement in this project, and Mr Adrian Webb for his technical help. The research was supported by the Royal Free Hospital School of Medicine through a Junior Research Fellowship, and by the Joint Research Board of Saint Bartholomew’s Hospital.

R E F E R E N C E S

BULLOUGH, W.S. (1968) The control of tissue growth. The Biological Basis of Medicine (Ed. by E. E. Bittar

CRILLY, R.G. (1972) Longitudinal overgrowth of chicken radius. J. Anaf. 112, 11. HALL-CRAGGS, E.C.B. & LAWRENCE, C.A. (1969) The effect of epiphyseal stapling on growth in length of

KEMBER, N.F. (1967) Cell survival and radiation damage in growth cartilage. Brit. J. Radiol. 40,496. KEMBER, N.F. (1972) Comparative patterns of cell division in epiphyseal cartilage plates in the rat. J. Anaf.

REIDY, J.A., LINGLEY, J.R., GALL, E.A. & BARR, J.S. (1947) The effect of roentgen irradiation on epiphyseal

and N. Bittar), Vol. I , chap. 9, p. 31 1 . Academic Press, London.

the rabbit’s tibia and femur. J. Bone J f Surg. SlB, 359.

111, 137.

growth. J. Bone Jt Surg. 29,853.