Cell Biology International Reports, Vol. 15, No. 2, February 7997 179

THE EFFECT OF BLUNT TRAUMA ON THE ULTRASTRl.JCTURE OF RATELLAR CHONDROCYTES IN RABBIT ARTICTJIAR CARTILAGE

A LIGHT AND ELECTRON MICROSCOPIC STUDY

Katsorchis Th., Kalogeromitros D. M.D., Makridimitri A.

Dr. Theodoros Katsorchis, University of Athens, Dept. of Biochemistry, Cell and Molecular Biology

and Genetics, Panepistimiopolis, Koupinia 157 01, Athens, Greece.

ABSTRACT

In order to determine the effects of mechanical injury upon articular cartilage, we caused trauma by a direct blow of 750 gr. weight on the flexed knees of rabbits. The joints were examined by light and electron micros- copy at two intervals after the injury.

In all specimens significant changes were observed in the cytoplasm of the chondrocytes. In addition, quantitative observations are reported to measure the observed degenerated ultrastructural changes.

INTRODUCTION

Cartilage is a form of connective tissue exclusively composed of chondrocytes which are involved in the production of the extra-cellular matrix. The typical ultrastructural features of cartilage cells and matrix have been examined, in the treatment of various kinds of joints diseases, over the last two decades (Lipshitz, et al., 1976; Mitchel, et al., 1989; Roberts, et al., 1986).

Both cartilage cells and the extracellular matrix dis- play considerable biological distortion, after either direct or indirect mechanical injury caused by trauma (Donohue, et al., 1983; Roberts, et al., 1988).

In addition, the histological and biochemical effects of steroids upon articular cartilage have been demonstrated in previous studies (Higuchi, et al., 1985; Koichiro, et al., 1981; Behrens, et al., 1975; Koehler, et al., 1974).

In the present study, we attempted to determine any alterations to the ultrastructure of cartilage cells

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caused by blunt mechanical injury upon the articular cartilage.

MWJ3RIALS ANDMETHODS

Source of articular cartilage: In all the experiments, fortyfive white New Zealand rabbits were used. The animals were housed in three different cages, in a ventilated room at a temperature of about 25°C. Trauma was induced in the fully flexed knee by a direct blow of 750 gr. weight from 1 m height, after general anaes- thesia of the animals with ether. Following the sac- rifice of the animals the articular cartilage of the patella and femoral condyles were studied in three separate groups of animals.

To provide comparative results, there was a control group of untraumatised animals.

Procedure for Tissue Preparation: The experimental and control animals were killed at the same time by decapit- ation whilst under light ether anaesthesia. Tissue specimens from the articular cartilage of the patella and femoral condyls were prefixed in sodium cacodylate buffer containing 3% glutaraldehyde and 2% paraform- aldehyde.

The specimens were then processed by the conventional procedures for light and electron microscopy.

Thin sections of 3-4~ in thickness were cut on a Sorvall MT-1 ultramicrotome and after staining with toluidine blue and sodium borate, were examined with a Leitz Ortholux Light Microscope.

Ultra thin sections were double stained with uranyl acetate and lead citrate and were examined with a Philips EM 200 Transmission Electron Microscope operating at 80 Kv.

In addition, quantitative data were obtained stereo- logically as described by Higuchi, et al., (1980).

RESULTS

Qualitative Observations

a) Control - Untreated Group: The specimens from the control group showed the typical characteristics of normal chondrocytes. As Figs la, 2a show the chond- rocytes were flat and eliptical in shape.

Cell Biology lnterna tional Reports, Vol. 7 5, No. 2, February 199 1 181

Fig.la. Light Micrographs. The Control group, normal chondrocytes are small, flat, and eliptical in shape (x1000). Fig.lb. Group - A: Chondrocytes appearing larger (single arrow) can be observed in the cytoplasm (x1000). Fig.lc. Group - B: Both the size of the cells and the quantity of the matachromatic substance is increased. The arrow shows the appearance of a vesicle in the region of the metachromatic substance (x1000).

182 Cell Biology International Reports, Vol. 15, No. 2, February 199 7

Cell Biology International Reports, Vol. 15, No. 2, February 1991 183

Fig.2a. Electron Micrograph. The Control Group, normal chondrocytes show typical features with large nucleus and various organelles (x11500). Fig.Zb. Group - A: The nucleus has migrated to the periphery of the cell. Note the appearance of electron-opaque material (glycogen) (arrow) and of electron lucent regions in the cytoplasm (x11500). Fig.2c. Group - B: The major part of the cytoplasm is occupied by the electron-opaque material and numerous vesicles. The normal organelles of the cell are hardly distinguishable in the periphery. The arrow points to a large lipid-like vesicle which con- tains amorphous material (x11500).

184 Cell Biology International Reports, Vol. 75, No. 2, February 1991

b) Group A - Experimental Animals: Thin sections from specimens obtained 15 days after injury showed few morphological changes in cells from the chondrocyte region. By light microscopy cells appeared larger compared to the control (Table III) and the presence of a metachromatic substance in the cytoplasm was noted as Fig lb shows. The metachromatic substance did not seem to occupy a specific region in the cytoplasm. Thinner sections examined in the (glycogen) electron microscope also showed the appearance of an electron opaque sub- stance and a number of electron lucent regions (Fig 2b and Tables I, II).

Cl Group B - Experimental Animals: Specimens obtained thirty days after the trauma showed significant differences in the chondrocytes from those obtained from the untreated group. The size and shape of the cells was altered considerably as shown in Fig lc.

Also the metachromatic substance and the vesicles observed by light microscopy were numerous and seemed to occupy the major part of the cytoplasm (Table III).

Ultra thin sections examined by electron microscopy showed that the cytoplasm was almost completely occupied by the electron opaque substance and, in addition, there are large lipid-like vesicles containing amorphous material (Fig. 2~).

Quantitative Observations

The above observations were followed by quantitative evaluations which were obtained stereologically as described by Higuchi, et al., (1980).

A transparent plastic sheet with a regular lattice of points was superimposed on to the micrographs. The number of points lying on the cellular organelles com- pared to the total number of points lying on the cell bodies represents the volume ratio of each examined structure in the chondrocytes (Tables I, II, III).

Table I: Ratio of the vesicles to the total cell surface area.

Groups Ratio (+. - sdl Increase (from Control).

CONTROL 7+, -1 GROUP A 10+, -1 65% GROUP B 30+, -1 76.6%

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Table II: Ratio of the electron opaque material WycogW.

Groups Ratio (+, - sdl Increase (from Control)

CONTROL 2+, -0.5 GROUP A g+, -0.5 77.7% GROUP B 14+, -0.5 85.7%

Table III: Alterations of the size of the cells measuring the maximum and the minimum diameter.

a) Minimum diameter

Groups Ratio (+, -sd) Increase (from Control)

CONTROL 14+, -2 GROUP A 21+, -2 41.6% GROUP B 24+, -2 41.6%

b) Maximum diameter

Groups Ratio (+, -sd)

CONTROL 22+, -2 GROUP A 23+, -2 GROUP B 26+, -2

Increase (from Control)

4.34% 15.38%

DISCUSSION

Injuries to joints are very common in humans and other mammalian species. There remains some confusion and contention on the structural response of articular cartilage to mechanical injury. The main purpose of this study was to determine the biological ultrastruct- ural effects on rabbit chondrocytes of blunt direct trauma.

It is obvious that mechanical injury to articular cartilage has varied considerably in its structural effects, depending on the nature of the injury (deep or superficial) and on the extent of the distortion that is created (Donohue, et al., 1983; Makela, et al., 1988).

Previous studies have examined the historical and ultra- structural changes observed in chondrocytes following either deep or superficial mechanical injury, mainly in human osteoarthritis (Roberts, et al., 1986; Spencer, et al., 1987).

The results obtained from our study suggest that the chondrocytes undergo progressive enlargement and degen-

186 Cell Biology International Reports, Vol. 15, No. 2, February 1991

eration following mechanical injury. This degenerative process was examined at two points after the trauma, namely 15 days and 30 days later.

One of the most important findings of the present study is the appearance of small electron lucent regions and large lipid vesicles in the general cytoplasm as observed two and four weeks after trauma.

Similar large vesicles were detected in the cytoplasm of chondrocytes as a result of systemic steroid administra- tion to articular cartilage (Bonucci, et al., 1984; Koehler, et al., 1974; Jacoby, et al., 1976). Most of these vesicles contain a glassy, homogeneous, amorphous material (Fig. 2b, 2c) whilst a few of them appear to be empty in the stained sections. Apparently they represent lipid-like vesicles.

It is notable that the amorphous material in the vesicles appears to be similar to the ground substance of which the extracellular matrix is composed. Based on the fact that the chondrocytes constitute the major countable component of the matrix, we assume that a structural resemblance may occur between the ground substance of the matrix and the material in the ves- icles. As a result we suggest that the ground sub- stance of the matrix, due to the mechanical injury, cannot be normally transported from the cells to the extracellular matrix, so it is concentrated in large amounts, and it is enclosed in vesicles in the cyto- plasm.

Furthermore, a metachromatic substance and electron opaque substance (glycogen) observed by both light and electron microscopy appeared to occupy the major part of the cytoplasm, four weeks after the trauma. Both the metachromatic and the electron opaque substances seem to exist in the normal chondrocytes but in very small quantities, (Tables I, II, III).

The increase in those substances observed mainly in the last stage (30 days) must be related to alterations in proteoglycan synthesis and metabolism as is suggested by i?vious studies (Higuchi, et al., 1980; Lipshitz, et

., 1975; Dhno, et al., 1988).

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Bonucci, E., Dearden, L. C., Rosier, J. R. (1984). Effects of Glycocorticoid Treatment on the Ultrast- ructure of Cartilage and Bone. Adv Exp. Med. Biol. 171: 269-279.

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Paper received 18.6.90. Revised paper accepted 1.12.90.