Osamu Horiuchi1), Ikuo Wada1), Nobuo Matsui1), Hong Jian Wang2), Yuji Asai2),
Takashi Yanagisono2), Eiji Yamada2), Daisuke Tamaki2), Toru Torii2),
Yoshio Mabuchi2) and Eisuke Sakuma2)
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

Key Words: Synovial Lining Cell, Rat, Development, Particle, Phagocytosis

Adress for correspondence: Osamu Horiuchi, Department of Orthopaedic Surgery, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan
Tel (+81) 52-853-8121; Fax (+81) 52-842-3210;

Morphological changes and functional maturation in the synovial lining cells of the rat knee joint were examined between day 20 and 30 after birth. Many oblique cells similar to fibroblasts were present in the synovial tissue on day 20. There were also a few undefined cells similar to macrophages. By day 30, two unique cell types could be distinguished: macrophage-like cells (type A cells) and fibroblast-like cells (type B cells). In addition, maturation of the selective phagocytotic uptake by the synovial cells using egg-lecithin coated polystyrene particles (240 nm) was demonstrated. On day 20, fibroblast-like cells were found phagocytizing both non-coated and lecithin-coated particles; however, macrophage-like cells showed minimal phagocytotic action. By day 30, well-defined type B cells could phagocytize only lecithin-coated particles, and non-coated particles were exclusively phagocytized by type A cells. The particles were also found in the capillary lumina of the 20 day-old rats. Joint cartilage debris and pressure changes during active joint motion might further lead to the maturation of the synovial lining cells. In addition particles found in the capillary lumina indicated that the capillaries were involved in the transfer of particles from the joint cavity.

It is well known that the mature synovial cells are composed of two main cell types based on their cytoplasmic characteristics (1-3). One is a macrophage-like cell (type A cell) and the other is a fibroblast-like cell (type B cell). It is generally accepted that type A cells have strong phagocytic potential, while type B cells have only minimal capabilites of phagocytizing foreign bodies. Type B cells are characterized by a highly developed rough endoplasmic reticulum with an expanded Golgi apparatus and are therefore thought to participate in protein synthesis. It is commonly believed that if the quantity of foreign particles is too great to be removed only by the type A cells, type B cells would work simultaneously with them to dispose of potentially harmful materials. We know of no studies in which mature type B cells showed selective phagocytosis of foreign substances. The present study, therefore, demonstrates the selective ability of the type B cells to take up egg-lecithin coated polystyrene particles that were injected into the intraarticular region of the rat knee joint.

Experimental condition;
All animal experiments were performed according to protocols determined by the Institutional Animal Care and Use Committee of Nagoya City University Medical School. The development of the knee joint synovium during the weaning period was studied in 20 and 30 day-old Wistar male rats (Japan SLC, Hamamatsu, Shizuoka, Japan). All rats were weaned from their mothers exactly on day 25, therefore we examined about the two distinctive periods; before and after the ablactation. A polystyrene particle suspension containing a 0.2% solid component was obtained from the Japan Synthetic Rubber Co. (Yokkaichi, Mie, Japan) and injected into the rat knee joints. The diameter of the latex particles was 240 nm and they had double spherical layers of polystyrene and methylmethacrylate. These particles were firstly applied to observe the uptake of foreign bodies by adult rat knee joint synovial cells (4). In addition, two types of particles were prepared for this investigation. A polystyrene particle suspension 1 ml was sonicated with or without 2 mg egg lecithin (Merck & Co., Inc.) for 4 min. using an ultrasonic sonicator to produce two types of particles, one coated by egg lecithin and the other uncoated. Both samples were resonicated just before injection to create a homogenous suspension.
The animals of each age were separated into three groups: Group A received saline (0.9% NaCl in distilled water, pH7.2) and served as the control, Group B was injected with non-coated particles and Group C received egg-lecithin coated particles. Each group consisted of 5 animals. All animals were anesthetized with ether and injected into their left knee joint cavity through the patella tendon with 0.05 ml of one of the three aforementioned solutions. The knees were then flexed and extended ten times. Right knee joints remained intact and served as controls. After awakening, the rats walked freely in their cages. One hour after the injections, the rats were killed by ether inhalation and the marginal zone of synovium adjacent to articular cartilage and underlying tissues was taken from the medial and lateral margins of the patellar area of each rat knee joints. The synovium adjacent to the patellar cartilage formed a fold (4), and this area is distinguished into areolar type synovium based on the standard classification (5).
Preparation for transmission electronmicroscopy;
These tissues 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 (pH7.4), and postfixed for 2.5 hr. in 1% osmium tetroxide buffered by 2% sucrose and 0.05 M sodium cacodylate (pH 7.4). After postfixation, the specimens were rinsed in ice-cold water for 5 min. and dehydrated in a graded series of ethanol. Following two 10 min. rinses in 100% ethanol, the specimens were immersed twice in absolute propylene oxide for 15 min. each and then embedded in epoxy resin (6). Thin sections were prepared, placed on copper grids, stained with uranyl acetate and lead citrate, and observed using Hitachi H-7000 transmission electron microscope.

Many oblique cells whose cytoplasmic appearance resembled fibroblasts were lying on the surface of joint cavity in the 20 day-old rats (Fig. 1). They displayed a well developed rough endoplasmic reticulum and an expanded Golgi apparatus. They were considered to be immature type B cells (IB). There were also a few undefined cells similar to macrophages (immature type A cells; IA) that had tubular invaginations of the plasma membrane, fine apical digitations and numerous mitochondria. The morphological features of the synovial cells had developed further by day 30, and almost all synovial cells could be distinguished into type A (A) or type B (B) cells (Fig. 2). Type A cells displayed numerous finger-like projections (arrows), many mitochondria, and an abundance of pinocytotic vesicles (arrow heads) and lysosomal bodies. By comparison, type B cells possessed a highly developed rough endoplasmic reticulum, an expanded Golgi apparatus, fewer mitochondria and many electron dense granules. Cellular contacts such as desmosomes, intermediate junctions and gap junctions were not found. Transitional type between type A and type B cells could never been found in this study.
After the intraarticular injection of saline or the particle suspension, no infiltration of inflammatory cells was observed in any of the experimental groups. Because the particles consisted of a cavity, this made it easier to distinguish them from intracellular organella under the transmission electron microscope. On day 20, immature type B cells phagocytized both non-coated (Fig. 3) and lecithin-coated particles (Fig. 4); however, immature type A cells showed minimal phagocytotic action (Fig. 5). Ten days later on day 30, type A cells had phagocytized particles of both types (Figs. 6 A,B), which was in contrast to type B cells that displayed selective phagocytotic action to only lecithin-coated particles (Fig. 7). A few particles could be found in the superficial capillary lumina in 20 day-old rats after intraarticular injection of lecithin-coated particles (Fig. 8). This superficial capillary possessed pericyte processes around the endotherial cells, so it could be distinguished not to be a peripheral lymphatic.

The synovial cells could be distinguished into two main types based on their cytoplasmic appearance. One was a macrophage-like cell (type A cell) whose macrophagic potential had been demonstrated using several types of substances, and the other was a fibroblast-like cell (type B cell) which is thought to play a role in protein synthesis. Type A cells are characterized by their fine cytoplasmic processes, abundant vacuoles (200 to 1500 nm in diameter) containing varying amounts of dense material and numerous tubular invaginations of the plasma membrane. Type A cells are thought to be main scavengers of the joint cavity. Type B cells are distinguished by a remarkably well developed rough endoplasmic reticulum, expanded Golgi apparatus and the presence of numerous vesicles containing dense material (150-350 nm in diameter). Barland et al. (1) reported these dense materials as being secretory granules.
There have been a number of studies describing the maturation and postnatal development of the synovial lining cells (2,3,7). According to Linck and Porte (2), the mouse metatarsophalangeal joints showed that cellular differentiation and proliferation of the synovial membrane occurred between 3 and 6 days after birth. Therefore, both type A and type B cells could already be distinguished by day 6. In the present study, we found immature type A and type B cells on day 20. On the other hand, almost all of the synovial cells had differentiated into type A or type B cells by day 30 and possessed cytoplasmic characteristics typical of the respective cell types. Thus, the morphological differentiation of these cells completed between days 20 and 30. The discrepancy between Linck and Porte (2) and the present study might be explained on the difference between mice and rats, or on the difference of joint position.
In addition, selective phagocytotic uptake by the two cell types was demonstrated using egg-lecithin coated polystyrene particles. Numerous investigators have studied the synovial membrane after intraarticular injection of various types of particles (8-12, 14). According to these investigators, type A cells took up large amounts of particles, both by engulfment and pinocytosis. In contrast, pinocytosis of smaller amounts of particles occurred by type B cells. They explained that if the quantity of particles was too great for the type A cells, then type B cells could be recruited to simultaneously dispose of the particles by phagocytosis.
The diameter of the particles in the present study was 240 nm. They had double spherical layers of polystyrene and methylmethacrylate and were firstly applied to examine the phagocytosis of particles by adult rat synovial cells (4). Because the particles had a cavity, it was easy to distinguish them from intracellular organella under the transmission electron microscope. In this study, there were only a few immature type A cells noted on day 20. These immature type A cells did not appear to have achieved functional differentiation, because they showed a little phagocytotic action. On the other hand, the immature type B cells had not developed the ability to differentially recognize the surface of the lecithin-coated particles and therefore phagocytized both types of particles. We could never found transitional type between type A and type B cells in the present study. Therefore, both type A and type B cells could already be distinguished by day 20 and differntiaton process took their courses independently in case of each cell type.
At 30 days of age, type B cells showed selective phagocytotic action to lecithin-coated particles, while type A cells phagocytized both non-coated and lecithin-coated particles. The latter did not seem to recognize the difference in the composition of the surface structures of the foreign bodies. Type A cells should play an important role in removing debris from the joint cavity; therefore, they appear to phagocytize all foreign substances in the joint cavity.
Type B cells are thought to recognize the surface of foreign substances such as egg-lecithin coated or non-coated before mounting a phagocytotic action. This ability is likely why they distinguished the membrane of the non-coated particles as being foreign, a phenomenon which appears to be absent in the type A cells. Thus, the ability to differentiate substances was established by type B cells before day 30. In the present study, very early stages of phagocytotic action of synovial cells was illustrated, and we present to our knowledge the first evidence demonstrating the selective phagocytotic action of the type B cells.
There was a weaning period between day 20 and 30 after birth. All rats began to readily move about their cages to secure their own food. The active movements of knee joints brought about wear of the cartilage and the deposition of debris into the knee joint cavity. The weight bearing process during movement also made a significant change in the pressure of the joint cavity. The debris and pressure changes might contribute to the maturation of the synovial lining cells and debris from cartilage wear would need to be removed from the joint cavity by some cellular transport mechanism. Moreover, this debris could be a trigger mechanism for the maturation of the synovial lining cells.
Lymphatic channels around joints are commonly understood to remove debris from the joint cavity. Several investigators have found changes in lymph nodes that drain the debris of large joints in experimental animals (13,14). In our study, we clearly demonstrated particles in the capillary lumina. This finding strongly indicates that the blood pathway co-contributes to debris removal from the joint cavity and should be studied more carefully when examining the mechanisms of debris removal in joints.

The authors thank Professor Damon C. Herbert (Department of Cellular and Strucural Biology, The Health Science Center at San Antonio, University of Texas) and Professor Tsuyoshi Soji (Department of Anatomy, Nagoya City University Medical School) for their scientific advice on this study.
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Figure Legends
Figure 1: A typical synovial membrane in 20 day-old rats. Almost all of the synovial cells were similar to fibloblasts with respect to their cytoplasmic morphology (immature type B cell; IB); a few macrophage-like cells were also found (immature type A cell; IA). Some fibloblasts (F) were also found.
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Figure 2: On day 30, almost all of the synovial lining cells could be distinguished as being either type A (A) or type B cells (B). Compared with figure 1, the lining cells seemed to be more densely arranged. Finger-like projections (arrows) and pinocytotic vesicles (arrow heads) of the type A cell could be found in this area.
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Figure 3: Synovial tissue in the 20 day-old animals after intraarticular injection of non-coated particles. Fibroblast-like cells phagocytized non-coated particles (arrow), while some particles were also scattered in the underlying fibrous zone (arrowheads).
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Figure 4: After the injection of lecithin-coated particles in the 20 day-old rat, an immature type B cells was observed phagocytizing lecithin-coated particles (arrows).
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Figure 5: On day 20, there were only a few macrophage-like cells which displayed phagocytotic action, particularly of the non-coated particles (arrows).
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Figure 6A: A type A cell (A) in a 30 day-old rat is illustrated phagocytizing non-coated particles (arrows). Note that the neighboring type B cells (B) show no phagocytotic action.
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Figure 6B: On day 30, one type A cell is shown phagocytizing lecithin-coated particles (arrows).
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Figure 7: In the synovial tissue of the 30 day-old rat that had received an intraarticular injection of lecithin-coated particles, type B cells display phagocytic action (arrows).
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Figure 8: A lecithin-coated particle with a hollow cavity (arrow) could be found within a capillary of the synovial tissue of a 20 day-old rat after intraarticular injection of lecithin-coated particles.
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