Growth of the mandibular condyle of the rat studied with tritiated thymidine

Arch. oral Bid. Vol.1 1, pp.493-500, 1966. Pergamon Press Ltd. Printed in Cit. Britain.

GROWTH OF THE MANDIBULAR CONDYLE OF THE RAT STUDIED WITH TRITIATED THYMTDINE

H. J. J. BLACKWOOD

Royal Dental Hospital of London, University of London, England

Summary-Immature female August strain rats were injected with tritiated thymidine and sacrificed at 4 hr and at 1, 3, 5 and 7 days following injection. Autoradiographs of coronal sections of the mandibular condyles were prepared and the number and distribution of the labelled cells within the condylar cartilages was recorded. Localiz- ation of labelled cells in the intermediate zone at 4 hr after injection indicates that this zone is the main growth layer of the cartilage. Cells from the intermediate zone differentiate to become chondrocytes which have a life span of 5-6 days before they are released into the medullary cavity. Renewal of cells in the articular zone was found to take place independently of the intermediate zone and it would appear that this zone acts only as an articular covering for the condyle and does not contribute to the growth of the condylar process. It is suggested that a reserve cell population may be present in the intermediate zone.

INTRODUCTION

THE CONDYLAR portion of the mandible in the rat as in most other mammalian species is developed in cartilage. This cartilage is derived from the blastema covering the dorsal extension of the growing mandible and is first seen in the rat at the 17-day insemination age (BHASKAR, 1953). At this stage it is separated from the developing squamotemporal bone by a condensation of mesodermal cells in which the joint spaces and articular disc of the mandibular joint will develop. The joint spaces are usually clearly defined by 19-day insemination age (BHASKAR, 1953).

The condylar cartilage forms an important growth centre for the mandibular ramus somewhat similar to the epiphyseal plate in a long bone but, from its position within the joint, it also provides the articular surface for the growing mandibular condyle.

Even from a relatively early stage of development this dual function is reflected in the structure and arrangement of the cells within the cartilage. The existence of three distinct cell zones is usually recognized (Fig. 1). There is a thin layer of flattened cells covering the cartilage which provides the articulating surface and deep to this is a band of densely packed cells usually referred to as the intermediate zone. This is regarded as the main progenitor or growth layer of the cartilage and mitoses can be observed much more frequently within this zone than in the other layers of the cartilage. The cells of this zone differentiate to become chondroblasts, or chondrocytes, and thereby give rise to the third layer or hypertrophic cartilage zone in which the cells and intercellular matrix undergo all the changes progressing to endochondral bone replacement of the cartilage.

493

494 H. J. J. Rl.AC-KWOOD

From a purely structural viewpoint it may be accepted that growth is provided

by the intermediate and hypertrophic cell zones of the cartilage and the articular zone serves as an articular covering for the cartilage. However, it cannot be deter-

mined by conventional histological methods whether the intermediate zone also acts

as a progenitor layer for the renewal of cells in the articular zone or whether the cells of this zone are capable of multiplication and renewal independently of the deeper zones. For this purpose tritiated thymidine was used in the present investig-

ation to study the pattern of cellular proliferation within the cell layers of the cartilage and to attempt to define more clearly the limits of each of the three cell zones des-

cribed above. Tritium-labelled thymidine specifically labels the desoxyribonucleic acid (DNA)

during chromosomal replication and owing to its accurate localization within the cell it may be detected within the cell nucleus and in succeeding cell divisions by high

resolution autoradiography. The techniques of tissue section autoradiography

( PELC, 1956; HERTZ, 1959) and the rationale for the study of cell populations using

tritium labelled thymidine have previously been described (HUGI~S cr ol., 195X;

PFI.C, I963 ; QLJASTFXR, 1963; WIMRER, 1963).

MATERIALS AND METHODS OF INVESTIGATION

Five hybrid August strain female rats of average weight 60-70 g (i.e. about 6-weeks-

old) which had been maintained on a standard stock diet were used for the experiment.

Each rat was given a single intravenous injection of 25 PC of tritiated thymidine in

0.5 ml of saline at a specific activity of 1.6-I -9 c/mMol and sacrificed at 4 hr and at

I, 3, 5 and 7 days after injection. The animals were decapitated, the pelt removed and the heads sectioned in the mid-line sagittal plane. The halved heads were fixed in 10 %

formol saline and then demineralized in 5 % formic acid. A block containing the whole

mandibular articulation was excised and embedded in paraffin wax. Sections through

the joint were cut in the coronal plane at 5-7 p in thickness and autoradiographs were prepared using Kodak A.R. 10 stripping film. The autoradiographs were exposed for 6 weeks at 4°C and following development were stained with Ehrlich’s haematoxy-

lin. Counts were made of the total number of labelled cells within each cartilage and

the position of labelled cells in each of the three zones of the cartilage was also re-

corded. The cells of the condylar cartilage, unlike those of an epiphyseal plate, are not evenly arranged in columns but in a section through the cartilage at right angles to the articular surface (Fig. 2) it was found that the articular zone was roughly five cells in depth, the intermediate approximately ten cells, and the hypertrophic zone also approximately ten cells, the greater depth of this zone being due to the large size of the hypertrophic cells. A cell could, therefore, occupy any of twenty-five levels

within the cartilage according to the scale shown in Fig. 2. Positions I to 5 represent the level within the articular zone, positions 6 to 15 the intermediate zone and 16 to 25 the levels within the hypertrophic zone,

GROWTH OF THE MANlXLNJLAR CONDYLE OF THE RAT STUIXEI’I WITH TRITlATED THYMIDINE 495

Counts of labelled cells were made at an initial magnification of 800 times using a squared eyepiece graticule and the position of the labelled cells recorded in relation to the scale described above. Ten sections of the cartilage from each animal were counted and the averaged figures from these counts were used to construct histograms in which the positions of the labelled cells were recorded on the abscissa and the number of labelled cells on the ordinate. At the medial and lateral margins of the cartilage the cell zones became narrower due to the curvature of the cartilage (Fig. 1) so these areas were excluded from the counts.

OBSERVATIONS

On microscopical examination the surface articular zone was perhaps the most easily and accurately identifiable zone as it did not vary much in thickness over the whole convexity of the cartilage. The junction of this zone with the intermediate zone was usually clear cut owing to the different character of the nuclei (Fig. 2). The nuclei of the intermediate zone (Fig. 3) were mainly spherical and densely packed together but the transition from the intermediate zone to the hypertrophic zone was difficult to define. Identification was based on the increasing size of the nuclei and the increasing separation of the cells due to the formation of intercellular matrix as the hypertrophic zone was approached (Fig. 4). The lower limit of the hypertrophic zone was accepted as the chondrocyte nearest to the medullary cavity occupying a lacuna, the walls of which were still intact and showed no evidence of resorption (Fig. 5). It was frequently noticed that as the chondrocytes approached the medullary cavity the nucleus tended to become vacuolated and the chromatin was displaced in a ring-like manner beneath the nuclear membrane. As a result when such a nucleus was positively labelled the grains appeared around the periphery of the nucleus (Fig. 6).

Tn all the autoradiographs counted the backgrounds were low and evidence of positive cell labelling was accepted as 5 or more grains per nucleus. The grain yields varied up to 40 or more grains per nucleus.

At 4 hr after injection by far the greatest number of labelled cells was found in the intermediate zone (Table 1) and these were concentrated mainly towards the centre of this zone (Fig. 7). A few labelled cells appeared in the adjacent area of the hypertrophic zone but no labelled cells were found throughout the remainder of this zone although numerous labelled osteoblasts or pre-osteoblasts could be seen on its deep aspect where the cartilage was being eroded by the vascular mesenchyme of the medullary cavity. In the articular zone not more than one or two labelled cells were found in each of the sections examined and these were located at varying levels within this zone (Fig. 8).

At 24 hr after injection the distribution of labelled cells was similar to the 4-hr period except that slightly more cells in the intermediate zone were labelled and these were localized mainly along the junction between the intermediate and hypertrophic zones. The level of labelling in the articular zone still remained very low and was spread throughout the zone (Fig. 8).

At 3 days qfter injection there was a marked spread of labelled cells from the intermediate zone into the adjacent part of the hypertrophic zone so that the level 01

496 H. J. J. BLACKWOOD

FIG. 7. Diagramatic outline of the condylar cartilage 4 hr after injection of Ha thymidine in which the positions of labelled cells are represented by dots and the cell zones marked. A, articular; I, intermediate; H, hypertrophic.

4 hours

--

10-l

1 day

3 days

5 days

, 0 4510 ARTICULAR IHTfRHfDIUE HYPERTROPHlC

ZONE 20NE ZOWE

Position of Cell

FIG. 8. Histograms showing the distribution of labelled cells in the condylar cartilage of the rat at 4 hr. 1, 3, 5 and 7 days after administration of tritiated thymidine.

GROWTHOFTHEMANDIBULARCONDYLEOFTHERATSTUDlEDWlTHTRlTIATEDTHYMIDlNE 491

labelled cells in this area equalled that of the intermediate zone (Table 1). However, the labelled cells did not extend through the full extent of the hypertrophic zone and their numbers fell sharply as the medullary cavity was approached. In this specimen practically no labelled cells were found in the articular zone (Fig. 8).

TABLE 1. TABLE SHOWING THE PERCENTAGE OF LABEL-

LED CELLS IN THE INTERMEDIATE AND HYPERTROPHIC

ZONES OF THE COPvDYLAR CARTILAGE

Time Labelled cells

Intermediate zone Hypertrophic zone

4 hr 1 dav

% % 3.6 0.17 3.8 064

3 da& 3.6 5 days 2.9 7 days 2.0 5.5

At 5 days after injection there was a much more uniform spread of labelled cells throughout the hypertrophic zone but they did not quite reach to the free edge of the cartilage plate bordering the medullary cavity. The percentage of labelled cells in the hypertrophic zone new exceeded the level in the intermediate zone (Table 1). The articular zone still showed very few labelled cells and these were present mostly at the medial and lateral edges of the cartilage and close to the articular surface (Fig. 8).

At 7 &+s after injection labelled cells were now spread throughout the full width of the hypertrophic zone and were present in relatively large numbers at the base of the cartilage plate adjacent to the medullary cavity. Labelled cells were still present throughout the intermediate zone but were reduced in number (Table 1). As in the previous specimens only a few labelled cells were found in the articular zone and there did not appear to be any tendency for labelled cells to spread to this zone from the intermediate zone (Fig. 7). It was also noticed in this specimen that occasionally some cells in the intermediate zone still showed a relatively high grain yield over their nuclei compared with neighbouring cells in the same area (Fig. 9).

DISCUSSION

In most rapidly proliferating cell systems uptake of tritiated thymidine may be accepted as an index of the proliferative activity of the tissue. In the present investigation tritiated thymidine has been used to define the proliferative cell zone, or zones, of the condylar cartilage and to study the movement of labelled cells within the growing cartilage. The marked localization of labelled cells in the intermediate zone observed at 4 hr and at 1 day following administration of thymidine (Fig. 8), therefore, confirms that this zone is the main proliferative cell compartment of the cartilage. Further, the concentration of labelled cells towards the centre of this zone suggests that maximum proliferative activity takes place within a very well defined area.

The upper limit of the intermediate zone, at its junction with the articular zone, is clearly marked by the abrupt decrease in labelled cells, and this line of demarcation

498 H. J. J. BLACKWOOD

remained constant throughout the observation period as shown in the later histograms (Fig. 8). The lower limit of this zone, however, is not so clearly defined as, at the 4-hr interval, labelled cells were also present in the adjacent layers of the hypertrophic zone, thereby indicating that some cell division may take place at this level in the cartilage. The presence of an occasional mitotic figure in this area of the cartilage gives proof to this assumption (Fig. 4).

The absence of labelled cells throughout the greater part of the hypertrophic zone at 4 hr after administration of thymidine suggests that this is a non-proliferative compartment populated by mature chondrocytes. This zone varied greatly in width being widest in the midline of the condyle and tapering rapidly towards either margin. For this reason accurate assessment of the relative position of labelled cells within this zone was difficult, and this was further complicated by the curvature of the cartilage. Also, towards the free margin of the cartilage plate the number of empty lacunae and their distance from the medullary cavity was extremely variable. The histograms showing the position of labelled cells in the hypertrophic zone are, therefore, subject to a certain degree of inaccuracy. However, it takes between 5 and 6 days for labelled cells to appear at the free edge of the cartilage plate adjacent to the medullary cavity. For the rats under examination in the present experiment the average width of the hypertrophic zone was 205 p so that the growth rate of the cartilage is approximately 35-40 p/day. This is an approximate estimation and is only about one third of the growth rate of the tibia1 epiphyseal plate as estimated by KEMBER (1960) for the same strain of rats of somewhat similar age group.

In contrast to the movement of labelled cells observed in the intermediate and hypertrophic zones it is interesting to note the persistently low level of cell labelling in the articular zone, and also the fact that during the total observation period there was no apparent movement of labelled cells from the intermediate zone into this zone. This would suggest that the cells of the articular zone are renewed independently of the deeper zones and possible also at a different rate.

A further possibility must be considered in respect of the small number of labelled cells in the articular zone and that is the availability of the label to this zone. WALKER and LERLOND (1958) and TONNA (1961) have reported a low yield of labelled cells in articular cartilage when tritiated thymidine was administered intravenously or intraperitoneally, but MANKIN (1962) has shown a relatively larger labelled cell population when the thymidine was injected directly into the joint cavity. MANKIN suggests that slow diffusion through the synovial membranes and cartilage matrix may be responsible for the lower yield of labelled cells when the intravenous and intraperitoneal routes are used. Whether this might have some effect on the labelling in the articular zone of the condylar cartilage is open to question but, on the other hand, it has been shown that tritiated thymidine is available to cells for 40 min or more following a single intravenous injection (STAROSCK et al., 1964). Tn a relatively small structure such as the condylar cartilage, this would appear to allow ample time for diffusion to take place so that the labelling observed in the articular zone should be a true assessment of the low proliferating activity of this zone.

It is interesting to compare the localization of labelled cells in the femoral articular

GKOWT~IO~THEMANDIUULAKCONDYLEO~THEKATS~UD~~DWLTH ~KI~IA~~DMYMIDINE 499

cartilage of the rabbit reported by MANKIN (1962) using the direct injection of tritiated thymidine into the knee joint with the findings of the present study. MANKIN described two distinct layers of labelled cells, one subjacent to the articular surface which he suggests contributes to the growth of this surface, and the other more deeply placed which contributes to the growth of the ossific nucleus. MANKIN does not give any definite figures for the numbers of labelled cells within each of these layers, but it would appear from his description that the mechanism of cell renewal and pattern of cell proliferation may be somewhat similar to that found in the condylar cartilage.

The observations of this study, therefore, offer fairly clear evidence for the existence of a separate articular zone of cells which functions primarily as a protective articular covering for the condyle. Renewal of cells in this zone is probably occasioned by the functional and growth requirements of the condyle, but there is no evidence to show that under normal conditions cells migrate to this zone from the underlying intermediate zone. It is possible, however, that some addition of cells may take place at the periphery from the periosteum to aid the increasing lateral dimensions of the cartilage during the growing period.

The problems inherent in the quantitative estimation and assessment of grain counts in autoradiographs have been discussed extensively (LEVI, 1957; LEVI and NIELSEN, 1959; PELC, 1957; HUGHJS et al., 1958; KOBURG, 1963) and this was not undertaken in the present study as there was found to be considerable variation in grain yields over nuclei in sections from different animals. It was noted, however, in the autoradiographs obtained from the animal sacrificed at 7 days, that occasionally some nuclei in the intermediate zone had much higher grain counts than others (Fig. 9) and the number of grains over such nuclei were comparable with those over the mature cartilage cells being shed at the free margin of the cartilage plate. This would suggest that not all cells within the intermediate zone divide regularly but that occasional cells may spend a longer time in their resting phase between mitoses than others. KEMBER (1960) reported a somewhat similar finding in the epiphyseal plate and postulated the existence of a reserve cell population at the top of the cartilage colums.

Finally, it must be mentioned that in the present study no attempt has been made to determine the cell cycle time, or generation time, of the cell population but it is hoped that this may be reported in a future study.

Acknowledgements-l should like to acknowledge the help and facilities olfered to me by Professor L. F. LAMERTON and Dr. N. KEMBER of the Institute of Cancer Research, Royal Cancer Hospital, London, during the initial preparation of this work and also Mr. J. MERCER for the photomicrographs, Miss JENNIFER MIDDLETON for the illustrations and Miss GILLIAN BAYLISS for her secretarial assistance.

Resume-Des Rates femelles impubi%es, d’une souche d’Aout, sont injectees avec de la thymidine tritiee et sacrifi&es apres 4 heures, 1 jour, 3 jours, 5 et 7 jours apres l’injection. Des autoradiographies de coupes des condyles mandibulaires sont effect&s. Le nombre et la distribution des cellules marquees dans les cartilages condyliens sont notes. La presence de cellules marquees dans la zone intermediaire, apres 4 heures

500 H. J. J. BLACKWOO

d’injcction, indiquc que cette zone est la couche de croissance principalc du cartilage. Des cellules de la zone intermediaire se differencient en chondrocytes qui ont une dure de vie de 5 a 6 jours, avant d’etre rejetQs dans la cavite medullaire. Le renouvcllement cellulaire dans la zone articulaire se fait independemment de la zone intermediaire et il apparait que cette zone ne joue qu’un rBle de recouvrement articulaire du condyle et ne contribue pas a la croissance du pro& condylien. II semble qu’une population cellulaire de reserve soit presente dans la zone intermediaire.

Zusammenfassung--Unreifenweiblichen Ratten vom August-Stamm wurde Tritium- markiertes Thymidin injiziert; die Tiere wurden 4 Stunden und 1, 3, 5 und 7 Tage nach der lnjektion get&et. Autoradiographien der coronalen Schnitte der mandibularen Condylen wurden angefertigt, und es wurde die Anzahl und Verteilung der markierten Zellen innerhalb der Condylarknorpel festgestellt. Die Lokalisation der markierten Zellen in der Intermediarzone 4 Stunden nach lnjektion weist daraufhin, dass diese Zone das Hauptwachstumszentrum des Knorpels darstellt. Zellen aus der Intermediar- zone ditIerenzieren zu Chondrozyten mit einer Lebenszeit von 5 bis 6 Tagen, bevor sie in den Markraum freigegeben werden. Die Erneuerung von Zellen in der Gelenkzone fand, wie festgestellt wurde, unabhangig von der Intermedilrzone statt, und es schien, dass diese Zone lediglich als eine Gelenkbedeckung fur den Condylus wirkt und nicht zum Wachstum des Gelenkfortsatzes beitrlgt. Es wird angenommen, dass eine Popu- lation von Reservezellen in der Intermedilrzone vorhanden sein dtirfte.

REFERENCES

BHASKAK, S. M. 1953. Growth of the rat mandible from 13 days insemination age to 30 days after birth. Atner. J. .Inuf. 92, l-53.

HARTZ, R. H. 1959. Methods to improve the performance of stripping emulsions. Lu6. Invrsr. 8, 71-75.

HUGHES, W. L., BOND, U. P., BRECH~K, G., CKONKIT~, E. P., PAIN~EK, R. B., QUASTELK, H. and SHERMAN, F. G. 1958. Cellular proliferation in the mouse as revealed by autoradiography with tritiated thymidine. Pror. Nut. Acad. Sri. 44, 476-483.

KEMBER, N. F. 1960. Cell division in endochondral ossification. J. Bone Jt Surg. 42B, 824839. KODUR~;, E. 1963. The use of grain counts in the study of cell proliferation. In: Cc/l Proliferatiurr

(Edited by LAMERTON, L. F. and FRY, R. J. M.) pp. 67-76. Blackwell, Oxford. LEVI, H. 1957. A discussion of recent advances towards quantitative autoradiography. Exp. Cell

Res., Suppl. 4, 207-221. LEVI, H. and NIELSEN, A. 1959. Quantitative evaluation of autoradiograms on the basis of track or

grain counting. Lob. Invest. 8, 82-91. MANKIN, H. J. 1962. Localization of triated thymidine in articular cartilage of rabbits. J. Bone

.It Surg. 44A, 682-688. I’ELC, S. R. 1956. The stripping film technique of autoradiography. fnl. J. uppl. Radiut. 1, 172-177. PELC, S. R. 1957. The quantitative aspects of autoradiography. Exp. Cell Res., Suppl. 4, 231-237. PELC, S. R. 1963. Incorporation of labelled precursors of DNA in non-dividing cells. In: CeN

Prohferution (Edited by LAMERTON, L. F. and FRY, R. J. M.) pp. 94-109. Blackwell, Oxford. QUASTLER, H. 1963. The analysis of cell population kinetics. In: Cell Prolifercrtion (Edited by

LAMERTON, L. F. and FRY, R. J. M.) pp. 18-34. Blackwell, Oxford. STAROSCK. R. N.. JENKINS. W. H. and MENDLESOHN. M. L. 1964. Availabilitv of tritiated thvmidinc

after ‘intravenous administration. Nature, Lond. 202, 456458. TONNA, E. A. 1961. The cellular complement of the skeletal system studied autoradiographically

with tritiated thymidine during growth and ageing. J. Biophys. Biochem. Cytol. 9, 813-824. WALKER. B. E. and LEBU)ND, C. P. 1958. Sites of nucleic acid synthesis in the mouse visualized by

radioautography after administration of Cl4 labelled ademine and thymidine. Exp. Cell Re.s. 14,510-531.

WIMBER, D. E. 1963. Methods for studying cell proliferation with emphasis on DNA labels. In: Cell Prohjerarion (Edited by LAMERTON, L. F. and FRY, R. J. M.) pp. 1-17. Blackwell, Oxford.

THYMIDINE

howing the le condylar

cells within ;in. x 270.

PLATE 1

H. J. J. BLACKWOOD

FIG. 3. Labelled nuclei in the intermediate zone at 4 hr after administration tritiated thymidine. Haematoxylin. 2 1000.

of

FIG. 4. The upper layer of the hypertrophic zone adjacent to the intermediate z( showing the increase in size of the cells and their separation by intercellular matrix, a one cell is shown in mitosis. Haematoxylin. x 1400.

3ne llS0

PLATE 2

GROWTH OF THE MANDIBULAR CONDYLE OF THE RAT STUDIED WITH TRITIATED THYMIDINE

aftei FIG. 5. Labelled chondrocyte at the free margin of the condylar cartilage 7 days

r administration of tritiated thymidine. Haematoxylin. x 1400.

PLATE 3

H. J. J. BLACKWOOD

FIG. 6. Labelled chondrocytes showing vacuola- tion of the nucleus and displacement of the label to the periphery of the nucleus. Haematoxylin. x 800.

FIG. 9. Variation in grain yields over nuclei in the intermediate zone 7 days after administration of tritiated thymidine. Haematoxylin. i: 1500.

PLATE 4

Documents

Growth of the mandibular condyle of the rat studied with tritiated thymidine