Elsevier

Bone

Volume 22, Issue 4, April 1998, Pages 331-339
Bone

Original Articles
Kinetic Studies on Epiphyseal Growth Cartilage in the Normal Mouse

Preliminary results of this study were presented at the 22nd European Symposium on Calcified Tissue, Vienna, Austria, 1991.
https://doi.org/10.1016/S8756-3282(97)00286-XGet rights and content

Abstract

The synthesis of DNA was studied in the proximal tibial growth plate of 25-day-old healthy NMRI mice by using the thymidine analog bromodeoxyuridine (BrdUrd), which is incorporated into cells in the S-phase. Such cells were found only in the upper three fifths of the morphologically defined proliferating zone. This zone was therefore subdivided into a functional proliferating zone (the S-phase zone) where most, if not all, chondrocytes proliferate, and a remaining maturation zone. The BrdUrd containing immunoreactive cells could then be followed at different intervals and they were found at the chondro-osseous junction after only 36 h. By using double-labeling with BrdUrd and iododeoxyuridine (IdUrd) the duration of cell cycle components could be estimated; that is, the time for DNA synthesis (S-phase), second gap and mitosis (G2 + M-phase), and remaining first gap (G1). We determined an S-phase time of 7.1 h and an average cell-cycle duration of 36 h. The G2 + M-phase was estimated as 3.5–4 h, leaving an average G1-phase time of 25 h, which probably varies considerably between chondrocytes. By combining these data with morphometrical data regarding distances between cells, we calculated a total growth rate of 9.0 μm/h. Of this rate, 80% was entirely related to the process of hypertrophy—that is, longitudinal expansion without any corresponding increase in cell number—and 75% was the result of processes outside the S-phase zone. Five percent of the growth was due to the expansion of cell distances within the S-phase zone. In this way longitudinal expansion can be studied at different levels in the growth plate and the data permit calculation of changes in volumes of the extracellular matrix. The largest increases in matrix volume occurred in the hypertrophic zone. These data may serve as a basis for further studies on matrix turnover in relation to growth.

Introduction

Growth cartilage is a form of highly specialized connective tissue with a characteristic morphological and biochemical composition. On strict light microscopic grounds, growth cartilage can be divided into four zones16, 18: (i) the resting zone; (ii) the proliferating zone; (iii) the hypertrophic zone; and (iv) the mineralizing zone. The resting zone is considered to contain inactive chondrocytes that serve as a pool of new cells for subsequent proliferation. In the proliferating zone, the cells undergo repeated mitotic divisions, thereby forming regular cell columns perpendicular to the growth plate. Proliferation is followed by cellular hypertrophy during which the cytoplasm increases in volume and appears more translucent and vacuolated at the level of both light and electron microscopy. In addition, the distance between cell centers increases due to the production of extracellular matrix components.

The intercolumnar septa of the hypertrophic zone then start to calcify, forming the border between the hypertrophic and mineralizing zones. Cells from the metaphysis then resorb parts of the mineralized matrix, and lay down new bone substance on the remaining mineralized cartilage, which thus serves as a backbone for metaphyseal bone formation. Because a continuous supply of mineralized cartilage is required for endochondral bone synthesis, this longitudinal expansion of cartilage is a rate-determining factor for growth of the long tubular bones.

The rate at which mineralized cartilage is formed, leading to longitudinal bone growth, depends on all the aforementioned events in the active growth plate. The first contribution to longitudinal bone growth takes place in the proliferating zone, where cells from the resting zone enter the proliferating zone and start to divide, forming longitudinally expanding columns. A second contribution consists of cellular hypertrophy in the hypertrophic zone,3, 10where an increase in cellular volume and the distance between cell centers further increases the height of the plate. As a third component, we must also consider the turnover of extracellular matrix, which is a process that parallels the cellular events. The matrix, which consists of glycoconjugates and proteins, is synthesized by chondrocytes and is rapidly deposited extracellularly. Well outside the cells, the matrix components are organized into macromolecular aggregates that constitute the organic part of the matrix. The matrix then persists, although with a changed composition,7, 11until it is finally incorporated into the metaphyseal bone and resorbed.

Measurements of the longitudinal growth rate have previously been performed indirectly, using the oxytetracyclin-labeling technique.3, 10, 21With the chondrocytes as reference points in the tissue, the expansion rate of the growth cartilage can be regarded as the net result of: (i) cell proliferation and cell disintegration; and (ii) cell hypertrophy. Direct estimates quantifying the expansion of the various zones seem to be sparse in the literature and the extent to which these processes contribute to the growth process in normal animals is not clear. The importance of hypertrophy has been well demonstrated both in normal animals1, 2, 3, 10and in the achondroplastic (cn/cn) mouse,[20]where premature mineralization obstructs normal hypertrophy. The complete cell cycle—that is, in the newly formed chondrocytes the first gap phase (G1) followed by DNA synthesis (S-phase), second gap (G2), and next mitosis (M-phase)—is only seen in the proliferating zone.

The aim of the present study was to measure the growth rate at any given level of the growth cartilage as well as to distinguish between the expansion rate related to cell proliferation and that due to cell hypertrophy. This was done by combining analyses of the distribution of S-phase cells and morphometric data. For this purpose, the distribution of cells in the S-phase were studied and their further progression in the tissue was followed over time. By combining morphometry and sequential immunochemical double-labeling, we measured the mean S-phase duration and expansion rates in the various epiphyseal zones.

Section snippets

Animals

Healthy NMRI mice were kept in plastic cages with free access to water and standard pelleted food where they were exposed to light for 12 h (6.00 a.m. to 6.00 p.m.) and to darkness for 12 h (6.00 p.m. to 6.00 a.m.). Only male animals were included. Animals were injected with labeling substances at a weight of 14–15 g corresponding to an age of 24–26 days, when the growth rate is maximal. Animals were killed by cervical dislocation at different times after the injections.

Reagents

The specific antibodies

Microspectrophotometry

The DNA ploidy patterns of the different zones in the growth cartilage are demonstrated in Fig. 1. All four zones were dominated by diploid cells. The tailing of the diploid peaks obtained was a result of measuring sliced nuclei (in both chondrocytes and reference cells). The resting, hypertrophic, and mineralizing zones contained no cells, indicating DNA synthesis and tetraploid cells were seen only in the upper part of the proliferating zone. These results indicate that DNA synthesis and

Acknowledgements

This study was supported by the Swedish Medical Research Council (Project No. 8274).

References (21)

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