INDEX
Effect of Joint Immobilization on Postnatal Development of Chondrocytes in Rat Knee Joints

Hiroshi Tomita1), Ikuo Wada1), Ikuo Sugimura1), Eisuke Sakuma2), Yoshio Mabuchi2)
and Nobuo Matsui1)
1)Department of Orthopedic Surgery, 2)The First Department of Anatomy
Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho,
Mizuho-ku, Nagoya, Aichi, 467-8601, Japan

Running Title: Postnatal Development of Rat Knee Joints
Key Words: Rat, Knee joint, Chondrocytes, Development, Immobilization
Address for correspondence: Hiroshi Tomita, Department of Orthopedic Surgery, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan
Tel (+81) 52-853-8236; Fax (+81) 52-842-0266;
E-mail: htomita@med.nagoya-cu.ac.jp


SUMMARY
It is generally accepted that immobilization of normal joints for a period of time causes the degenerative changes in the articular cartilage of mature and immature joints. We studied the alteration of cartilage in the rat knee joint between day 20 and 30 after birth by using light and electron microscopy. Twenty day-old Wistar strain male rats were used in this study. The rats were divided into two groups; those receiving immobilization and controls. In the immobilization group, the right knees of 20-day-old rats were fixed in extended position with a rigid fiberglass cast. After immobilization for five days, these rats were killed and the right knee joints were observed. In the control group, rats aged 20, 25 and 30 days were killed and the right knee joints were observed. The articular cartilage in the immobilization group showed increased cellular population, and there were flattened or elliptical shaped cells with insufficient cytoplasm. Atypical cells were found in the most superficial area of the cartilage. Their cytoplasm partly projected from the articular surface and they had some circular mitochondria with preserved cristae and some slightly dilated rough endoplasmic reticulum. Their configurations suggested active movement up through the superficial zone to the articular surface. We believe these phenomena are the survival reactions of immature articular cartilage under abnormal conditions.

INTRODUCTION
Immobilization of normal joints for a period of time generally causes the degenerative alterations in their articular cartilage. Several authors have described the histological and biomechanical changes in experimental immobilization (1~11). Long-term immobilization of animal joints causes progressive histological changes that include the proliferation of connective and synovial tissues, fibrous adhesions to articular surfaces, cartilage erosion and cartilage necrosis with subchondoral bone alterations. From the evidence of earlier studies, immobilization from four to seven weeks was necessary for articular cartilage alterations to become evident (1, 2). In more recent studies, histological or electron microscopic changes were observed two weeks after immobilization and the qualitative changes were observed only one week after immobilization (4, 5). Furthermore, the initial changes of the articular cartilage occur within the first few days after the onset of joint immobilization (3, 6). In these studies, only adult and young adult animals were investigated, however, and there has yet been no clear evidence of the alterations in the articular cartilage of immature animals following short term joint immobilization. In the present study, we investigated the early changes in the patellar articular cartilage of neonatal rat knee joints after short-term immobilization. Because the initial changes are mostly evident within the superficial zone of the articular cartilage, we observed the changes of chondrocytes in the superficial articular cartilage by using light and transmission electron microscopes.

MATERIALS AND METHODS
All experiments using animals were performed according to the animal handling regulations of Nagoya City University. Twenty Wistar strain male rats were used in this study. All animals were 20 day-old at the start of the study. The rats were divided into two groups; those receiving immobilization and controls. In the immobilization group, the right knees of five rats were immobilized in extended position with a rigid fiberglass cast from the trunk to the foot under mild ether anesthesia. Care was taken not to wrap the hind limb so tightly as to seriously impair circulation. All of the rats were housed in standard cages and moved freely during five days. At the age of 25 days, the rats of the immobilization group were killed and the patellae of the right knee joints were removed. These patellae served for observations by transmission electron microscopy (TEM). In the control group, five rats aged 20, 25 and 30 days were killed and the right knee joints served for the observations. Two kinds of specimens were made in this group. One was for the light microscopic observations, the other was for the TEM.

Preparation for Light Microscopy (LM):
For the light microscopic observations, the patellae with the femoral condyles were used. The tissues were fixed overnight in 4% paraformaldehyde buffered by phosphate (pH 7.4) at 4. Decalcification by EDTA was used only in the case of 30 day-old rats that began to manifest endochondral ossification of the patella, in order to prevent distortion of the tissue in the process of sectioning. After dehydration through graded series of ethanol, specimens were embedded in Paraplast embedding media (Sigma Chem. Co, St. Louis, MO, USA). After trimming of blocks, axial sections were made in 2m thicknesses. The sections were flattened on the surface of a thermostat-regulated water-bath at 37 and dried in a desiccator at 40. They were stained with hematoxylin and eosin.

Preparation for Transmission Electron Microscopy (TEM):
For the TEM studies, the patellae of the right knee joints were fixed immediately by immersion for 2 hr. in an ice-cold solution of 2.5% glutaraldehyde and 2% sucrose in 0.05 M cacodylate buffer (pH 7.4), and post fixed for 2.5 hr. in 1% osmium tetroxide buffered by 2% sucrose and 0.05 M sodium cacodylate (pH 7.4). After post fixation, the specimens were rinsed in ice-cold water for 5 min. and dehydrated in a graded series of ethanol. Following two rinses in 100% ethanol for 10 min. each, the specimens were immersed twice in absolute propylene oxide for 15 min. each and then embedded in epoxy resin (12). Ultra thin sections were prepared, placed on copper grids, stained with uranyl acetate and lead citrate, and observed using a Hitachi H-7000 transmission electron microscope.

RESULTS
Control Groups
Twenty Day-Old Rats:
The patella of a 20 day-old rat was wholly composed of hyaline cartilage, and showed high cellularity (Fig.1). Under TEM observation, the patellar articular cartilage of the 20 day-old showed a rough surface with hollows in places. Profiles of the cells in the superficial region were elliptical and these cells were arranged radical to the joint surface (Fig.2). In this stage, the attributes of the superficial zone were different from those of mature articular cartilage. The articular cartilage near the surface showed high cellularity with sparse extra cellular matrix, and the cells were arranged randomly. The cells in the superficial zone were elliptical and had rich rough endoplasmic reticulum in scant cytoplasm. Their plasma membrane demonstrated many cytoplasmic processes. The upper part of the middle zone consisted of rounded chondrocytes whose cytoplasms were more abundant (Fig.3).
Twenty-five Day-Old Rats:
The patellae articular cartilage in 25 day-old rats were still composed solely of hyaline cartilage with lowered cellularity compared to the 20 day-old. The cells in the superficial region were closer together than those in the deeper region (Fig.4). Under TEM observation, most of the cells in the superficial region were flat in shape. Elliptical shaped cells were occasionally seen in this region. The articular surface of the patella was smoother than that of 20 day-old rats (Fig.5). The cellularity in the superficial zone was still high. This zone consisted of small flattened cells arranged parallel to the joint surface, indicating further maturation of the articular cartilage. The cells in the upper part of the middle zone showed elliptical shape and their cytoplasms were more abundant (Fig.6).
Thirty Day-Old Rats:
In 30 day-old, formation of the articular cartilage of the patella was in progress as enchondral ossification proceeded (Fig.7). In this stage, the cellularity had decreased in comparison to 20 or 25 day-old. Although the cells in the superficial region were still closer together than those in the deeper region, the four distinct zones as seen in an adult articular cartilage were clearly recognized. The superficial zone consisted of small-flattened cells with scant cytoplasm arranged parallel to the joint surface (Fig.8). The upper part of the middle zone consisted of elliptical or rounded cells with proliferating cytoplasm, and the extra cellular matrix was more abundant.

Immobilization Group
Twenty-five Day-Old Rats (immobilized for five days):
The articular cartilage in this group showed high cellular population (Fig.9). Under the electron microscope, there were flattened or elliptical shaped cells with inadequate cytoplasm (Fig.10). In contrast to the 25 day control, the cellularity of the superficial region was rather high and the chondrocytes displayed flatter morphology. Moreover, this articular cartilage showed a rough surface with hollows similar to the 20 day-old control (Fig.11). There were small and thin cells adhering to the surface in the hollows. The nuclei of these cells were flattened and pyknotic. The nuclei had a smooth contour and a clear nuclear membrane, though the contents showed uniformly low electron density. The ratio of the cytoplasm to the nucleus was small. Mitochondria of these cells had barely preserved cristae, and rough endoplasmic reticulum with polyribosomes were widely dilated. These cells lacked a distinct plasma membrane on the side of the joint space. The chondrocytes of the superficial zone showed elongated or flattened morphology with many branched cytoplsmic processes. These features are consistent with those of chondroblasts. A most marked observation at this stage was the occurrence of atypical cells found in the most superficial area of cartilage. Their cytoplasm projected partly from the articular surface and they had circular mitochondria with preserved cristae and slightly dilated rough endoplasmic reticulum. They had fewer cytoplasmic processes and were morphologically different from the surrounding chondrocytes of the superficial zone. Their configuration resembled that of the degenerative cells on the surface in the hollows (Fig.12, 13).

DISCUSSION
It has been discussed in previous studies that the structural characteristics of articular cartilage vary with age (13~24). Surface irregularity, cell density, morphological features of chondrocytes and others are different between immature and mature articular cartilage. Immature articular cartilage displays a rough articular surface with many hollows and sparse extracellular matrix with high density of randomly arranged cells. These chondrocytes in the superficial zone show an elliptical shape and have many rough endoplasmic reticulums in the scant cytoplasm. On the other hand, in the mature articular cartilage, the cellularity decreases and four distinct zones become clearly recognized. The superficial zone consists of small flattened cells which are arranged parallel to the smooth joint surface. The extracellular matrix is more abundant. Cells in the superficial zone change their morphological features and numbers with the course of maturation. The articular cartilage of the rat knee joint is still considered to be immature at 20 day-old. After the rats are weaned at approximately 25 days, activity of their hind limb becomes more brisk and the maturation of their articular cartilage prgresses. As a result, the articular cartilage of 30 day-old rats is well matured. Therefore, 20 day-old rats are considered to be suitable for study about the influences of joint immobilization on the immature articular cartilage.
In a sequence of these changes, both intrinsic and extrinsic factors synchronously affect the maturation of articular cartilage. The intrinsic factors are cell development and production of extra cellular matrix. The extrinsic factors are physiological joint motion and weight bearing. Several authors have reported that restriction of physiological joint motion and weight bearing by joint immobilization cause the degenerative alteration of articular cartilage in mature articular cartilage (1~11). Whether joint immobilization will induce similar degenerative alteration in immature articular cartilage has not been clarified. In our experiments, the articular cartilage of the immobilization group showed high cellular population, and there were flattened or elliptical shaped cells with inadequate cytoplasm similar to those in the 20 day-old control rats. Particularly, in contrast to the 25-day control, the cellularity of the superficial region was rather high and flattened shaped chondrocytes were increased in the immobilization group rats. Moreover, this articular cartilage showed a rough surface with hollows. These observations suggest that joint immobilization interrupted or delayed articular cartilage maturation. In addition, there were small, thin degenerated cells adhering to the surface in the hollows. Occasionally, some of these cells protruded from the articular surface to the joint cavity. These atypical cells were seen in the most superficial area of the cartilage. They were morphologically different from the nearby chondrocytes of the superficial zone. Their configuration indicated active movement up through the superficial zone to the articular surface. Cole et al (25) reported analogous cells of the femoral head articular cartilage in neonatal (from 5 to 15 day old) rats; the cells resting on the articular surface and the cells projecting from the articular surface. He suggested that as some of the chondrocytes in the superficial zone of the neonatal articular cartilage began to degenerate, they moved up or were moved up toward the articular surface and finally shed into the joint space. After the chondrocytes detached from the articular surface, the depressions in the articular surface represented vacated chondrocyte lacunae. This shedding of chondrocytes was rarely seen after two weeks of age. Nevertheless, we observed cells similar to these shedding chondrocytes even in 25 day-old rats which received immobilization.
Our observations can be explained as follows. Restriction of physiological joint motion and weight bearing in immature rats interrupts the maturation of the articular cartilage. As a result, chondrocytes of the superficial region remain immature, and this alteration makes the 25 day-old articular cartilage of the immobilization group resemble the 20 day-old articular cartilage of the control group (Fig.14). Moreover, these chondrocytes of the superficial zone undergo further disruption of their maturation process. They actively move up through the superficial zone toward the articular surface, begin to degenerate and are finally shed into the joint space like those seen in neonatal articular cartilage. We consider these phenomena to be survival actions of immature articular cartilage under abnormal conditions. The high cell content per unit volume of cartilage under immobilized conditions does not allow the getting of an adequate territory to maintain normal cellular activity. The shedding of the chondrocytes may be a response to these conditions with the goal of normalization of cartilage cellularity and hence a return to normal cartilage development.
In conclusion, immobilization of the immature joint dose not causes the degenerative changes in articular cartilage that have been observed in studies with mature joints. In immature articular cartilage, immobilization causes some chondrocytes of the superficial zone to actively move up toward the articular surface and finally be shed into the joint space as seen in the neonatal articular cartilage. These phenomena are necessary to prevent the gross degeneration of immature articular cartilage under abnormal conditions. Joint immobilization not only retards development of articular cartilage, but also causes retrogression of chondrocytes development.

FIGURE LEGEND
Figure 1. Light photomicrograph of the 20 day-old rat patella.
The cells in the superficial region show elliptical shape and are arranged radical to the joint surface.
X 400
Figure 2. Electron micrograph of the 20 day-old rat patellar articular surface show some hollows in places.
X 2,000
Figure 3. The cells in the superficial zone are elliptical shaped, and have rich rough endoplasmic reticulum in scant cytoplasm. Their plasma membrane has many cytoplasmic processes.
X 5,000
Figure 4. Light photomicrograph of the 25 day-old rat patella.
The cellularity is less than in 20 day-old. The cells in the superficial region are closer together than those in the deeper region.
X 400
Figure 5. Electron micrograph of the 25 day-old rat patella. The articular surface is smoother than that of 20 day-old rats, and the hollows are not as evident. Most of the cells in the superficial region are flat in shape.
X 1,500
Figure 6. The superficial zone consists of small flattened cells which are arranged parallel to the joint surface. The cells of the upper part of the middle zone show elliptical shape and their cytoplasms are more abundant.
X 2,000
Figure 7. Electron micrograph of the 30 day-old rat patella. The cellularity has decreased
in comparison to 20 or 25 day old.
X 2,000
Figure 8. The superficial zone consists of small flattened cells with scant cytoplasm.
They are arranged parallel to the joint surface.
X 5,000
Figure 9. Light photomicrograph of the 25 day-old rat patella after immobilization for five days.
X 400
Figure 10. Electron micrograph of the 25 day-old rat patella after immobilization for five days.
In contrast to the 25-day control, the superficial region shows high cellularity and flat shaped chondrocytes are increased. The surface of this articular cartilage is rough with hollows similar to the 20 day-old control. An atypical cell is in the most superficial area (arrow).
X 1,000
Figure 11. A cell adhering to the surface in the hollow.
The plasma membranes on the side of the joint space are not distinct. These cells seem to be degenerating chondrocytes with damaged plasma membranes on the side of the joint space and poorly defined organelles.
X 10,000
Figure 12 and 13. Atypical cells in the most superficial region.
Their cytoplasm partly projects from the articular surface and they have circular mitochondria with preserved cristae and slightly dilated rough endoplasmic reticulum.
12 and 13: X15,000
Figure 14. A summerizsed pictures of 20 (a), 25 (b) and immobilized 25 (c) day-old.
Immature profile of articular cartilage in immobilized group is clear.
a, b, and c : X3000

REFERENCES
1. Sood SC. A study of the effects of experimental immobilisation on rabbit articular cartilage. J Anat 1971; 108:497-507.
2. Finsterbush A, Friedman B. Early changes in immobilized rabbits knee joints: a light and electron microscopic study. Clin Orthop 1973; 92:305-319.
3. Troyer H. The effect of short-term immobilization on the rabbit knee joint cartilage. A histochemical study. Clin Orthop 1975;107:249-257.
4. Langenskiod A, Michelsson JE, Videman T. Osteoarthritis of the knee in the rabbit produced by immobilization. Attempts to achieve a reproducible model for studies on pathogenesis and therapy. Acta Orthop Scand 1979; 50:1-14.
5. Candolin T, Videman T. Surface changes in the articular cartilage of rabbit knee during immobilization. A scanning electron microscopic study of experimental osteoarthritis. Acta Pathol Microbiol Scand 1980; 88:291-297.
6. Jurvelin J, Helminen HJ, Lauritsalo S, Kiviranta I, Saamanen AM, Paukkonen K, Tammi M. Influences of joint immobilization and running exercise on articular cartilage surfaces of young rabbits. A semi quantitative stereomicroscopic and scanning electron microscopic study. Acta Anat 1985;122: 62-68.
7. Paukkonen K, Jurvelin J, Helminen HJ. Effects of immobilization on the articular cartilage in young rabbits. A quantitative light microscopic stereological study. Clin Orthop 1986; 206:270-280.
8. Kiviranta I, Tammi M, Jurvelin J, Arokoski J, Saamanen AM, Helminen HJ. Articular cartilage thickness and glycosaminoglycan distribution in the young canine knee joint after remobilization of the immobilized limb. J Orthop Res 1994; 12:161-167.
9. Setton LA, Mow VC, Muller FJ, Pita JC, Howell DS. Mechanical behavior and biochemical composition of canine knee cartilage following periods of joint disuse and disuse with remobilization. Osteoarthritis Cartilage 1997 ; 5:1-16.
10. Fu LL, Maffulli N, Yip KM, Chan KM Articular cartilage lesions of the knee following immobilisation or destabilisation for 6 or 12 weeks in rabbits. Clin Rheumatol 1998;17: 227-233.
11. Haapala J, Arokoski J, Pirttimaki J, Lyyra T, Jurvelin J, Tammi M, Helminen HJ, Kiviranta I. Incomplete restoration of immobilization induced softening of young beagle knee articular cartilage after 50-week remobilization. Int J Sports Med 2000; 21:76-81.
12. Luft JH. Improvement in epoxy resin embedding methods. J Biophys Biochem Cytol 1961; 9: 409-414.
13. Whillis J. The development of synovial joints. J Anat 1940; 74: 277-283.
14. Andersen H. Histochemical studies on the histogenesis of the knee joint and superior tibio-fibular joint in human foetuses. Acta Anat 1961; 46: 279-303.
15. Andersen H. Histochemistry and development of the human shoulder and acromioclavicular joints with particular reference to the early development of the clavicle. Acta Anat 1963; 55: 124-165.
16. Mitrovic DR. Development of the metatarsophalangeal joint of the chick embryo: morphological, ultrastructural and histochemical studies. Am J Anat 1977; 150: 333-348.
17. Mitrovic DR. Development of the diarthrodial joints in the rat embryo. Am J Anat 1978; 151: 475-486.
18. Mitrovic DR. Development of the articular cavity in paralyzed chick embryos and in chick embryo limb buds cultured on chorioallantoic membranes. Acta Anat 1982; 113: 313-324.
19. Drachman DB, Sokoloff L. The role of movement in embryonic joint development. Develpment Biol 1966; 14:401-420.
20. Craig FM, Baylis MT, Bentley G, Archer CW. A role for hyaluronan in joint development. J Anat 1990; 171: 17-23.
21. Archer CW, Morrison H, Pitsillides AA. Cellular aspects of the development of diarthrodial joints and articular cartilage. J Anat 1994; 184: 447-456.
22. Edwards JC, Wilkinson LS, Jones HM, Soothill P, Henderson KJ, Worral JG, Pitsillides AA. The formation of human synovial joint cavities: a possible role for hyaluronan and CD44 in altered interzone cohesion. J Anat 1994; 185: 355-367.
23. Pitsillides AA, Archer CW, Prehm P, Bayliss MT, Edwards JCW. Alterations in hyaluronan synthesis during developing joint cavitation. J Histochem Cytochem 1995; 43: 263-273.
24. Bland YS, Ashhurst DE. Development and ageing of the articular cartilage of the rabbit knee joint: distribution of the fibrillar collagens. Anat Embryol 1996; 194: 607-619.
25. Cole MB Jr, Narine KR, Ellinger J. Morphological evidence of the shedding of chondrocytes from the articular surface in neonatal rats: relationship to the interlacunar network. Anat Rec 1983; 206: 439-446.


Nagoya City University
Medical School