Hyaluronidase

ABSTRACT

Purpose. To study the cause and mechanism of joint degeneration in osteoarthritis, through histopathological and ultrastructural-histochemical experiments on the articular cartilage of the knees of the C57 black mouse.

Methods. 192 C57 black mice and a control group of 64 C57BL/6J mice were used in this study. The left and right knee articular capsules of the joints were removed and stained. Each articular cartilage sample was examined and osteoarthritic changes were assessed using a transmission electron microscope. The severity of osteoarthritis in the knee joint cartilage of C57 black mice was histologically assessed using a classification system described by Okabe, based on Maier's system.

Results. The incidence and the severity of osteoarthritis gradually increased with age; the incidence increased from 20% at 2 months to 80% at 16 months. Irreversible changes appeared at an advanced stage, and the process of degeneration was quite similar to that in human osteoarthritis. Through transmission electron microscopy, we observed poorly developed Golgi apparatus, markedly increased intracellular microfilaments, decreased proteoglycan granules, and broken collagen networks in all stages of osteoarthritis. By contrast, Golgi apparatus and other organelles were well developed in histologically normal mice of all ages. Proteoglycan granules, which mainly consisted of keratan sulphate, were observed; collagen networks were maintained.

Conclusion. Disturbed protein transport and sugar synthesis in chondrocytes, caused by the deficient development of the Golgi apparatus, could result in degenerative changes in articular cartilage. The structure and function of the matrix were maintained mainly because of the continued presence of keratan sulphate.

Key words: cartilage, articular; histocytochemistry; osteoarthritis; ultrastructure

INTRODUCTION

Based on its structural and nutritional characteristics, articular cartilage is governed by a complex mechanism of cartilage metabolism and proteoglycan synthesis. Because of its association with mechanical movements, articular cartilage is more likely to be affected by endogenous and exogenous factors than other motor-related tissues.

Primary osteoarthritis (OA) is a chronic, progressive, and non-inflammatory articular disease that often affects weight-bearing joints, and is characterised by a mixture of degenerative and proliferative lesions in articular cartilage. The main symptoms associated with OA are degeneration and repair of the articular cartilage.

Any combination of mechanical, nutritional, biochemical, and immunological factors could be involved in the formation of the initial lesions; however, causative factors of OA have yet to be identified.1 Furthermore, it remains unclear whether subsequent degeneration is caused by changes in the cartilage matrix or the chondrocytes.

Age-induced cartilage changes have been associated with OA2; however, no conclusive evidence has been obtained. Radiological and anatomicopathological analyses have shown well-conserved cartilage in some older people, and severely degenerated cartilage in some young people. As a result, we concluded that factors other than age are more important in causing the development of OA.

Various experimental models have been developed regarding the onset and pathology of human OA. In most of these models, secondary OA is induced by artificial ligament or cartilage defects, but few studies have been conducted to investigate the factors associated with OA and its initial pathological features. When investigating the initial pathological features of OA, experimental models must closely resemble human OA: onset mechanisms need to be comparable, the progression of initial lesions must be gradual, and arthritic changes need to be continuous and progressive. The C57 black mouse is an appropriate animal model for human OA research because degeneration occurs more frequently in adulthood and progresses gradually with age. This animal OA model was first reported by Silberberg and Silberberg3 in 1960. A genetic mutation with recessive Mendelian inheritance makes these mice susceptible to OA. OA of these mice occurs spontaneously during adulthood and progresses gradually, sharing numerous pathological similarities with human OA. Okabe4 has carefully investigated degenerative changes in these mice by light microscopy.

In recent years, studies using C57 black mice have been conducted in various laboratories, but none have closely investigated the incidence and degenerative process of spontaneous osteoarthritis.

In our study, transmission electron microscopy (TEM) and TEM histochemical methods were used to observe the ultrastructure of the articular cartilage in C57 black mice. We aim to elucidate the factors that trigger OA during the initial degenerative process and how this process differs from simple ageing of the cartilage.

MATERIALS AND METHODS

C57 black mice were raised and bred by mating between siblings at the Tokyo Medical University Animal Center according to the criteria established by Good Laboratory Practice.5192 C57 black mice (6 males and 6 females per month; at 1-16 months old) and a control group of 64 C57BL/6J mice (2 males and 2 females per month; at 1-16 months old) were used in this study conducted between July 2000 and March 2003.

The left and right knee joints, including the subchondral bone, were removed from both groups of mice. The articular capsule of one joint was placed under a stereomicroscope. It was then cut and separated into femoral and tibial sides by desmotomy. Having been removed as much subchondral bone as possible using a scalpel, the joint was fixed in 4% paraformaldehyde and 1% glutaraldehyde (0.1 M phosphate buffer solution, pH 7.4) at 4°C for 24 hours, and then demineralised using 4% EDTA (ethylenediaminetetraacetic acid) containing 0.2 M sucrose at 4°C for 2 weeks. Each sample was then sliced along the sagittal plane, washed in 0.1 M phosphate buffer solution overnight (pH 7.4), fixed in 2% osmium tetroxide (phosphate buffer solution, pH 7.4) at 4°C for 2 hours, dehydrated using an increased series of ethanol, and embedded in Epon812 (TAAB Laboratories Equipment Ltd, Berks, United Kingdom).

Five super-thin sagittal sections (about 60 nm) of the medial compartment of the tibia for each knee were prepared using a Reichert ultramicrotome (Leica Microsystem GmbH, Wien, Germany). They were then double stained using uranyl acetate and lead acetate, and then phosphotungstic acid. A transmission electron microscope (Hitachi H-600A, Tokyo, Japan) was used to observe each articular cartilage sample and to assess osteoarthritic changes in 3 layers-the superficial layer, intermediate layer, and deep layer (calcified cartilage)-under an accelerating voltage of 75 kV and direct magnifications of 1800 to 48 000 times. Five cells in each of the 3 layers were analysed for each of the 5 sections of each knee. In addition to the super-thin sections, 1-µm sections were also examined under a light microscope after staining by toluidine blue (pH 4.1).

The other joint was stained using ruthenium red according to Luffs6 methods, and subjected to various glycosaminoglycan (GAG) digestive enzyme treatments using chondroitinase ABC (Chase ABC), chondroitinase AC (Chase AC) [both from Seikagaku Kogyo, Tokyo, Japan], testicular hyaluronidase (THase), streptomyces hyaluronidase (SHase), and keratanase (Kase) [all from SIGMA-Aldrich Co, St. Louis (MO), US] under predetermined conditions (Table 1). The results of electron-microscopic histochemical analyses were investigated.

The severity of OA in the knee joint cartilage of C57 black mice was histologically assessed after haematoxylin-eosin staining, using a classification system that was described by Okabe4 and based on Maier's system (Table 2). The severity of OA was classified into 5 stages as follows: stage 0, normal; stage I, damage in the superficial cartilage layer; stage II, damage down to the tidemark; stage III, damage in all cartilage layers; and stage IV, damage down to the bone. To assess initial changes in articular cartilage, stage I was divided further, based on the degree of reduced metachromasia after pH 4.1 toluidine blue staining. Stage I-1 corresponded to reduced metachromasia in the superficial layer; stage I-2, reduced metachromasia in the cell region of the non-calcified cartilage layers; stage I-3, reduced metachromasia in the intercellular region; and stage I-4, reduced metachromasia in all cartilage layers.

RESULTS

Confirmed by light microscope, the incidence of OA (stage I-1 and above) among C57 black mice increased with age from 25% at 2 months to 80% at 16 months. Likewise, the severity of OA also increased with age. Nonetheless, approximately 25% of the C57 black mice were still at stage 0 even at 16 months.

Stage 0 mice were classified as the physiologically normal ageing group ('ageing group'). Mice with conserved cartilage layers (stages 1-1 to 1-4) were classified as the OA-related group ('OA group'). Electron-microscopic findings between these 2 groups are compared below.

Electron-microscopic findings of the ageing group at 1 to 4 months

Chondrocyte: in the superficial layer, cells and nuclei were both flat and spindle-shaped; the long axis was aligned parallel to the articular surface. Although few cytoplasmic processes were observed, organelles such as rough endoplasmic reticula and mitochondria were relatively well developed (Fig. 1a). In the intermediate layer, a variety of cell shapes were present, but cytoplasmic processes or nuclear envelope indentations were not discernible. These chondrocytes were cytoplasm-rich, containing well-developed, largely tubular, rough endoplasmic reticula and numerous tubular and spherical mitochondria (Fig. 1b). Fine granules, which appeared to be proteoglycans, were detected in the pericellular matrix by ruthenium red staining. In the deep layer, the lacuna was enlarged, and the lateral wall partially exhibited a lamellar structure. The cytoplasm of deep layer cells tended to be atrophie.

Matrix fibre structure: in the superficial layer, fibres between 15 to 20 nm in diameter were densely aligned parallel to the articular surface. The lamina splendens was poorly developed. In the intermediate and deep layers, a low-density, 3-dimensional mesh of 30 to 60 nm fibres with poorly developed, periodic crossstriation patterns was observed. Among these fibres, numerous ruthenium red-positive granules were visible. Digestion of these granules was excessive with Chase ABC, Chase AC, and THase, but was incomplete with Kase and SHase, which suggested the granules were composed mostly of chondroitin sulphate.

Electron-microscopic findings of the ageing group at 5 to 11 months

Chondrocyte: although cells in the superficial layer were slightly rounded, few morphological changes were seen. In the intermediate layer, lacuna enlargement and cytoplasmic processes were slightly more discernable, but no marked changes were observed in intra-cellular organelles. Rough endoplasmic reticula and mitochondria were well developed, and free ribosomes were dense. In the pericellular matrix, numerous ruthenium red-positive granules were observed. In the deep layer, cytoplasmic atrophy was noticeable.

Matrix fibre structure: the fibre arrangement and morphology of each layer was comparable to that observed in the mice that were 1 to 4 months old, but the diameters of these fibres were slightly greater.

Electron-microscopic findings of the ageing group at 12 to 16 months

Chondrocyte: the cell density of the superficial layer in each section was slightly lower. Although intracellular organelles were generally underdeveloped, there was no clear decrease in cellularity. In the intermediate layer, increased cytoplasmic processes and nuclear envelope indentations, as well as nuclear pyknosis were observed. Rough endoplasmic reticula were expanded and fused; mitochondria were slightly enlarged; and Golgi apparatus were mostly intact (Fig. 2a). In the pericellular matrix, ruthenium redpositive granules were relatively well conserved. Histochemical analysis showed that digestion was incomplete with Chase ABC, Chase AC, THase, and SHase, but was excessive with Kase (Figs. 2b, 2c, and 2d). These results suggested that keratan sulphate was the main component of these granules. In the deep layer, cellular amorphism was further advanced.

Matrix fibre structure: the diameter of fibres in the superficial layer was 20 to 30 ran, whereas that in the intermediate and deep layers was 40 to 80 nm. Periodic cross-striation was partially observed, but the 3-dimensional mesh work of these fibres was conserved. Digestion of the ruthenium red-positive granules seen among the fibres was excessive with Kase, but was incomplete with the other enzymes, suggesting that keratan sulphate was the main component of matrix GAG granules in the intercellular region. Additionally, the number of matrix vacuoles was high and they concentrated around the tidemark; however, no tidemark thickening or stratification was seen.

Electron-microscopic findings of the OA group at stage I-1

Chondrocyte: the density of flat spindle-shaped cells in the superficial layer was lower, but no marked morphological changes were noticed. The development of cytoplasmic organelles was normal, and little difference was noted when compared with the ageing group (stage 0). In the intermediate layer, numerous mitochondria and well-developed, tubular, rough endoplasmic reticula were seen. Nuclear envelope indentations were not obvious. Although relatively clear nucleoli were visualised, Golgi apparatus development was poor. In the pericellular matrix, ruthenium red-positive granules were relatively well conserved. In the deep layer of the subchondral bone side, a lamellar structure stained deeply by ruthenium red was seen on the lateral wall of the lacuna.

Matrix fibre structure: although the arrangement of fibres in the superficial layer was not markedly disturbed, the lamina splendens was either thin or absent. The 3-dimensional meshwork was maintained in the intermediate and deep layers, but when compared with the ageing group, the diameter of fibres was generally greater, at 50 to 90 nm. Periodic crossstriation was slightly clearer. In addition, a large number of ruthenium red-positive granules were seen among these fibres.

Electron-microscopic findings of the OA group at stage 1-2

Chondrocyte: the density of cells in the superficial layer was even lower, and most cells were slightly rounded. Additionally, cytoplasmic processes and lacuna enlargement were more pronounced. In the intermediate layer, cluster formation was noticeable and surrounded by a large number of granule deposits (matrix vacuoles). In the chondrocyte cytoplasm, saccular dilatation of rough endoplasmic reticula and glycogen granules were noticeable (Fig. 3). In the deep layer, the ring structure of chondrocytes was even more pronounced.

Matrix fibre structure: the morphology of fibres in stage 1-2 was comparable to that in stage 1-1.

Electron-microscopic findings of the OA group at stage 1-3

Chondrocyte: in the superficial layer, cells exhibited nuclear pyknosis and poorly developed organelles. In the intermediate layer, saccular dilatation of rough endoplasmic reticula was more pronounced than that in stage 1-2, and the reticula lumens were filled with fine protein-like granules. Golgi apparatus were poorly developed, and perinuclear intermediate filaments were diffusely distributed in the cytoplasm (Figs. 4a and 4b). In the deep layer, concentrated organelles and cytoplasmic matrices were seen in the majority of cells.

Matrix fibre structure: compared to stages 1-1 and 1-2, the fibre diameters in all layers were greater, but the 3-dimensional meshwork was mostly intact.

Electron-microscopic findings of the OA group at stage 1-4

Chondrocyte: even in the superficial layer, the lacuna was well defined and enlarged, and increased cytoplasmic processes and elliptical cells were clearly observed. Nuclear pyknosis and reduced organelles were even more pronounced than those in the earlier stages. In the intermediate layer, largely irregular cell morphology, nuclear envelope indentations, nuclear pyknosis, and increased heterochromatin were observed (Fig. 5). Rarefaction of the cytoplasmic matrix, vacuolisation of rough endoplasmic reticula, and atrophy of other organelles were also more pronounced. In the pericellular matrix, there were a reduced number of ruthenium red-positive fine granules, which were digested excessively with Chase ABC, Chase AC, and THase, but incompletely with Kase and SHase. Therefore, these granules were thought to consist mostly of chondroitin sulphate. Cells in the deep layer were even more amorphic, and the lamellar structure on the lateral wall of the lacuna was thick circumferentially. When this structure was subjected to various digestive enzyme treatments, the results were comparable to those for the pericellular matrix fine granules, suggesting that this structure was also mainly composed of chondroitin sulphate.

Matrix fibre structure: in all layers, fibres were thicker and some were fragmented into smaller pieces. The density of these fibres, the periodic cross-striation patterns, and the number of ruthenium red-positive granules, were low. Particularly in the deep layer, the meshwork was partially absent, and some fibres were aligned perpendicularly to the articular surface.

DISCUSSION

Cartilage degeneration in OA cannot be attributed to a single factor. Although various factors were involved, cartilage degeneration has been explained in terms of matrix destruction caused by increased enzymatic activity, which was due to various stresses on chondrocytes and the matrix.7 Although different factors have been associated with initial osteoarthritic changes, not many studies have specifically analysed the chondrocytes themselves. The changes in the cartilage of the ageing group have been examined as aetiologic factors. Because they are pathologically similar to those of the OA group, the initial lesions caused by OA were characteristically similar to those caused by the ageing process. C57 black mice were used because they spontaneously develop osteoarthritis in a fashion comparable to humans. In the hope of elucidating the degenerative process and factors associated with OA, we conducted ultrastructural and histopathological analyses to investigate the cells and matrix of articular cartilage, as well as electron-microscopic and histochemical analyses to clarify the differences between OA- and age-induced changes of cartilage.

Age-induced changes in articular cartilage

Several electron-microscopic studies on age-induced changes in articular cartilage have been conducted.8-12 Because all of these studies examined OA-related physiological changes, the normal, physiological ageing changes in articular cartilage had not been studied. In the present study, we used a light microscope to conduct histochemical analyses on articular cartilage, to identify joints unaffected by OA, and then examined these joints electron-microscopically to ascertain physiologically normal, age-induced changes in articular cartilage.

Our study showed that under the normal ageing process, the density of chondrocytes did not decrease with age. Although mild nuclear envelope indentations and nuclear pyknosis were seen, the karyoplasm was generally even, and nucleoli were well defined. Intranuclear activities, such as ribosome synthesis, were also intact. In the cytoplasm of chondrocytes, the development of rough endoplasmic reticula was favourable. Although partial saccular enlargement was seen after 12 months, active protein synthesis was confirmed. Golgi apparatus was well developed, and even in older mice, the shrinkage of the Golgi apparatus was not seen, suggesting that saccharide synthesis and addition functioned smoothly. Some mitochondria were large, but they mostly remained intact. Also, the concentration or rarefaction of the cytoplasmic matrix was not marked, and free ribosome degeneration was not indicated. Therefore, the energy supply for the chondrocytes was intact. As age increased, fibrils appeared around the nuclear margins, but the surrounding organelles were not compressed. As a result, protein metabolism and synthesis in the cytoplasm of chondrocytes was conserved relatively well in older mice. The diameter of collagen fibres increased gradually and periodic cross-striation became more pronounced with age, but the arrangement of these fibres (especially the 3-dimensional meshwork in the intermediate and deep layers) was not disturbed. In addition, age-induced tidemark stratification or thickening described by Lane and Bullough,13 and fibres aligning perpendicular to the articular surface around the tidemark described by Weiss14 were not observed. Furthermore, electron-microscopic and histochemical analyses demonstrated that the decrease in the absolute volume of GAG in the pericellular matrix and intercellular matrix was low, and that GAG composed of kera'-an sulphate remained mostly intact. These findings suggest that when articular cartilage ages normally, the matrix fibre structure is well conserved, partially aided by the presence of GAG, which is mainly made of keratan sulphate.

Under normal ageing processes, the physiological activity of chondrocytes gradually decreases to lower protein and GAG syntheses. This causes a change in the composition of matrix GAG, but the matrix fibre structure remains intact because GAG, which mainly consists of keratan sulphate, is still present.

Initial changes in osteoarthritic articular cartilage

Several detailed electron-microscopic studies have been conducted on osteoarthritic articular cartilage by Little et al.15 in 1958 and Weiss14 in 1973. However, most of these studies examined mature OA, and most light-microscopic studies examined relatively advanced OA. In recent years, few electron-microscopic studies on the relationship between age-induced changes and OA-induced changes have been conducted.10,14 A conclusive relationship has not been established. In this study, we identified joints with early osteoarthritic changes and examined them by electron microscopy to clarify the onset mechanism of OA at the ultrastructural level, and to identify the differences between changes caused by the normal ageing process and by OA.

The results showed that as OA advanced, the density of chondrocytes decreased, especially in the intermediate layer. Additionally, nuclear envelope indentations, advanced nuclear pyknosis, and increased heterochromatin were seen, suggestive of reduced intranuclear activities (eg ribosome synthesis). In the cytoplasm of chondrocytes, saccular dilatation of rough endoplasmic reticula was seen, and these reticula were filled with proteins, particularly in the intermediate layer, which indicates active protein synthesis.16 Conversely, according to Ogawa and Nagano,17 this suggests impaired protein transportation from the rough endoplasmic reticula to the Golgi apparatus. The present study showed impaired Golgi apparatus development, thus supporting the latter hypothesis. The impaired development of the Golgi apparatus is a unique characteristic of all early osteoarthritic changes. It may interfere with saccharide synthesis, introduction, and sulphation, as well as the addition of saccharides to proteins synthesised in the rough endoplasmic reticula; this will ultimately affect matrix GAG. Nonetheless, more detailed observations and studies are needed to identify the factors involved in impaired Golgi apparatus development. Several hypotheses have been advanced to explain the accumulation of intermediate filaments in the cytoplasm of chondrocytes in stages 1-3 and 1-4. These include: excessive protein pool,16 degeneration of intracellular components,18 cytoskeleton, and areas of excessive collagen representing incomplete collagen. Kouri et al.19,20 reported that cytoskeleton was abundant for clonal chondrocytes in human OA cartilage, and that unlike normal chondrocytes, the modification of cytoskeleton was observed. As a result, the intermediate filaments seen in the present study appeared to be cytoskeleton. In any case, these changes, together with impaired Golgi apparatus development, are thought to result in impaired protein transport, or impaired GAG synthesis and secretion. Furthermore, findings such as nuclear pyknosis, poorly developed nucleoli, and the concentration, rarefaction, and atrophy of cytoplasmic free ribosomes, all suggested that chondrocyte activity was reduced. Unlike the normal ageing process, various structural changes such as fragmented collagen fibres, reduced fibre density, fibre rarefaction, destruction of the 3-dimensional meshwork, and perpendicular arrangement were observed as OA advanced. As reported by Anderson and Sajdera,21 a marked decrease in GAG among fibres is thought to play a large role in these structural changes. The circumferential accumulation of chondroitin sulphate on the lateral wall of the lacuna is also believed to correlate with the decrease in GAG among fibres. Furthermore, increased numbers of pericellular matrix vacuoles were seen after stage 1-2, but as reported by Fujiwara22 and Kagoya and Furuhashi,23 trypsin-like proteases in the matrix vacuoles are activated when these vacuoles rupture. The subsequent release of proteases decomposes and eliminates the surrounding matrix GAG.

In summary, impaired Golgi apparatus development mainly affects chondrocytes in the intermediate layer. It can be further complicated by intracellular fibril accumulation. It follows that protein transportation, saccharide synthesis, and saccharide addition become hindered, which then impair extracellular GAG secretion and matrix diffusion. Furthermore, destruction of proteoglycans is attributable to increased matrix vacuoles, which leads to the collapse of the fibre structure (Fig. 6).

The articular cartilage changes of C57 black mice caused by OA and the normal ageing process are shown in Tables 3 and 4, respectively. The results show that cartilage degeneration did not always start in the superficial layer. Initial degeneration was mostly seen in the intermediate layer, and prior to matrix degeneration, cytoplasmic ultrastructural changes and impaired Golgi apparatus development were observed. As a result, reduced GAG synthesis in chondrocytes led to secondary destruction of the matrix structure.

Under the physiologically normal ageing process, intracellular ultrastructure remained relatively intact throughout the study period. Although vacuolisation of rough endoplasmic reticula, accumulation of fibrils, nuclear pyknosis, and formation of fat droplets have been associated with normal ageing; they were more often seen in osteoarthritic joints. The diameters of matrix collagen fibres were slightly greater, but the fibre structure was maintained, and perpendicular arrangement and tidemark stratification were hardly seen in the deep layer. This suggested that some findings associated with the normal ageing process could actually represent OA. The results of our electron-microscopic analyses on abundant GAG-which was mainly made of keratan sulphate and aided the maintenance of the collagen fibre structure-supported age-induced changes in GAG composition, which have been documented by biochemical, lightmicroscopic, and histochemical studies.24-26

CONCLUSION

We investigated whether C57 black mice are suitable animal models for human osteoarthritis and conducted electron-microscopic analyses to identify onset factors for OA, its initial degenerative processes, and the differences between cartilage changes caused by OA and by normal ageing.

Under the physiologically normal ageing process, reduced protein synthesis (caused by gradual reductions in chondrocyte activities) was observed, but the matrix structure remained mostly intact, which is partially due to the retention of keratan sulphate.

Under OA changes, both protein synthesis and saccharide synthesis and addition were impaired, particularly in the intermediate layer. Meanwhile, destruction of the matrix structure was also observed. Impaired Golgi apparatus development was identified as an initial change in the ultrastructure of chondrocytes in the intermediate layer, but its cause could not be identified.

These findings suggest that osteoarthritic changes are not necessarily caused by the physiologically normal ageing process, but in combination with other factors.

The results of the present study can help clarify the factors involved in human primary OA and its initial degenerative process.

ACKNOWLEDGEMENT

The authors would like to thank Prof J Patrick Barron of the International Medical Communications Center of Tokyo Medical University for reviewing this manuscript.

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K Yamamoto, T Shishido, T Masaoka, A lmakiire

Department of Orthopedic Surgery, Tokyo Medical University, Tokyo, Japan

Address Correspondence and reprint requests to: Dr Kengo Yamamoto, Assistant Professor, Department of Orthopedic Surgery, Tokyo Medical University, 6-7-1 Nishishinkuku, Shinjuku-ku, Tokyo, 160-0023, Japan. E-mail: kengo-y@tkg.att.ne.jp

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