THE ULTRASTRUCTURE OF EQUINE SYNOVIAL MEMBRANE

HorIcyD.,F. Tichy: The Ultrastructure of Equine SynovialMembrane. ActavetBmo,I995,64:225-229. The ultrastructure of synovial membrane was studied in two adult stallions of the English thorougbred horse. Samples were collected from 2 astragalar and 2 knee joints of stallions. The equine synovial membrane was composed of two cell types, A and B, and the synovial matrix which extended into subsynovial tissue. Synoviocytes,located singly, were usually covered with synovial matrix; they were in contact with the joint cavity only occasionally. A-cells showed a structure generally similar to the mammalian cells of this type but they were flatter and had no cytoplasmic projections. The cytoplasm of B-cells was free of secretory granules and lacked fibrils passing through the plasma membrane. The synovial matrix of the surface layer showed numerous dense bodies. The occurrence of thick bundles of aperiodic filaments which were several Jlll1long and 0.2-0.3 Jlll1 thick was an unusual finding. Ultrastructure, synovial matrix, synovial A cells, B cells, horse The synovial membrane plays a key role in both physiology and pathology of synovial joints. This important finding was made fairly early and since then the structure of synovial membrane has been the subject of many investigations. First studies dealt with synovial membranes in experimental animals and later in humans. The present-day knowledge on its microscopic and submicroscopic structure as well as biochemical and immunochemical data on both normal and diseased joint are based on investigations carried out in a wide variety of mammalian and avian species by researchers in various biological fields (for review see Horky 1990, 1991). Observations of numerous authors have shown that the synovial membranes of all species so far studied are composed of two types of synovial cells, first classified by Barland et al. (1962), i. e., A orM(macrophage-like) andB orF (fibroblastlike) cells. A-cells are similar in structure to histiocytes. Their phagocytic capacity has been demonstrated in many mammalian species (Cutlip and Cheville 1973; Leach et al. 1988; Fabry 1989). B-cells, also found in most of the mammalian species, are characterised by well developed granular endoplasmic reticulum, an extensive Golgi apparatus and the presence of secretory granules. Occasionally these cells are also referred to as S (secretory) cells. In addition to Bar 1 an d et al. (1962), they have been described by other authors, most recently by Meek et al. (1991), Worrall (1991), Levandon and Stein (1992) andPasquali et al. (1991) in normal joints and in those affected by various pathological conditions. In horses, information on synovial membrane is scarce. The first more detailed observations were presented by Johanson and Rejno (1976) who compared synovial membranes of healthy joints with those of joints affected with intraarticular fractures of osteochondrosis dissecans. Equine synovial membrane is also mentioned in the work of Gangel (1984) and Hilbert et al. (1984) dealing with the properties of synovial fluid. These facts and our long-term involvement in studies on synovial membranes (Horky 1990, 1991, 1993) have led to a decision to investigate this structure in the horse and thus contribute to information on this articular component in this mammalian species.

The synovial membrane plays a key role in both physiology and pathology of synovial joints.This important finding was made fairly early and since then the structure of synovial membrane has been the subject of many investigations.First studies dealt with synovial membranes in experimental animals and later in humans.The present-day knowledge on its microscopic and submicroscopic structure as well as biochemical and immunochemical data on both normal and diseased joint are based on investigations carried out in a wide variety of mammalian and avian species by researchers in various biological fields (for review see Horky 1990Horky , 1991)).Observations of numerous authors have shown that the synovial membranes of all species so far studied are composed of two types of synovial cells, first classified by Barland et al. (1962), i. e., A orM(macrophage-like) andB orF (fibroblastlike) cells.A-cells are similar in structure to histiocytes.Their phagocytic capacity has been demonstrated in many mammalian species (Cutlip and Cheville 1973;Leach et al. 1988;Fabry 1989).B-cells, also found in most of the mammalian species, are characterised by well developed granular endoplasmic reticulum, an extensive Golgi apparatus and the presence of secretory granules.Occasionally these cells are also referred to as S (secretory) cells.In addition to Bar 1 an d et al. (1962), they have been described by other authors, most recently by Meek et al. (1991), Worrall (1991), Levandon andStein (1992) andPasquali et al. (1991) in normal joints and in those affected by various pathological conditions.
In horses, information on synovial membrane is scarce.The first more detailed observations were presented by Johanson and Rejno (1976) who compared synovial membranes of healthy joints with those of joints affected with intraarticular fractures of osteochondrosis dissecans.Equine synovial membrane is also mentioned in the work of Gangel (1984) and Hilbert et al. (1984) dealing with the properties of synovial fluid.These facts and our long-term involvement in studies on synovial membranes (Horky 1990(Horky , 1991(Horky , 1993) ) have led to a decision to investigate this structure in the horse and thus contribute to information on this articular component in this mammalian species.

Materials and Methods
Samples of synovial membrane were collected from 2 knee joint of two English thoroughbred stallions aged 2 years and from 2 astragalar joints of a stallion 8 years old.The samples of synovial membranes with attached subsynovial tissue were dissected, when immersed in fixation medium, into strips (l x 1 x 2-3 mm) and immediately placed in 300 mmol/L glutaraldehyde in 0.1 M phosphate buffer at pH 7.4, first for 60 min and then, in a fresh solution, for further 180 min.They were subsequently rinsed with 3 exchanges of 0.1 M phosphate buffer at pH 7.4.This was followed by fixation with 40 mmol/L OsO 4 in phosphate buffer at pH 7.4 for 15 and 45 min.Dehydration, immersion and embedding in Durcupan ACM were carried out using standard methods, polymerisation took 3 days in the oven at 60 •C.Ultrathin sections were cut in an LKB ultramicrotome, stained with lead citrate according to Reynolds or by a method combining 1 % uranyl acetate with lead citrate.They were examined and photographed in a Tesla BS 500 electron microscope.Semithin sections for light microscopy observations were stained with methylene blue and Azure II.

Ultrastructure of synoviocytes
The equine synovial membrane was covered by two, occasionally three, layers of cells.These layers were covered by synovial matrix (Figs,1,2,4).Deeper in the membrane, the matrix gradually became continuous with subsynovial tissue.Only small surface areas were not covered with the matrix and cells were in direct contact with the joint cavity (Fig. 3).The synovial membrane was not separated from subsynovial tissue by a basement membrane.

A-cells
In the equine synovial membrane, A-cells were found in lower numbers than B-type cells.They were most frequently located near the synovial surface, presenting as individual cells (Fig. 1).They differed in shape from the corresponding cells of other mammalian species; they were oval or elongated and measured about 8-10 x 2-4 Jlm.
The nucleus was elongated or rod-like, with large, marked karyosomes situated at the nuclear envelope (Fig. 1).The nucleolus, if present, was of a reticular type.The zonula nucleum limitans was narrow, forming only a thin layer attached to the inner membrane of the nuclear envelope (Fig. 1).
The cytoplasm was present in a small amount as a narrow lining around the nucleus (Fig. 1).The granular endoplasmic reticulum presented as occasional short, flattened cisternae.The Golgi apparatus took up only a small region of cytoplasm and, if present in the section viewed, it showed its usual appearance.Mitochondria were small with only few cristae.Areas rich in cytoplasm contained smooth, electron transparent vesicles.Close to the cell membrane, there were pinocytotic vesicles.Lysosomes, centrioles or cytoplasmic filaments were not observed.
The cell membrane provided a smooth cover to almost all the surface.Occasionally, it extended into the surrounding matrix as short filopodia (Fig. 1).

B-cells
B-type cells were more frequent than A-cells and occurred as individual cells (Fig. 2) or in pairs (Fig. 5) either under or on the synovial surface (Fig. 3).In the latter case, they were located in folds of the membrane, were not covered by intercellular substance and were in direct contact with the joint cavity (Fig. 3).
The nucleus either was oval-shaped or had an irregular outline due to deep invaginations created by the nuclear envelope.Chromatin appearance was similar to that in A-cells.
The cytoplasm was present in larger amounts than in A-cells (Figs 2, 3, 5) with granular endoplasmic reticulum presenting as numerous narrow cisternae varying in length or as dilated vesicles up to 0.5 Ilm in size (Figs 2, 3) with fmely granulated contents.Agranular endoplasmic reticulum was seen as numerous smooth, electron transparent vesicles, 0.2-0.3Jlm long.In addition to these structures, B cell cytoplasm, in areas close to the cell membrane, showed smooth vesicles, about 0.8 Jlm, with granular content which were considered transport vacuoles (Figs 2, 4) and also smaller vesicles, 0.4-0.5 Jlm, filled either with dark homogeneous material or with structures resembling autophagic vacuoles or pseudomyelin structures (Fig. 2) .
. The Golgi apparatus was similar in appearance to that in A-cells.Its cisternae sequestered smooth vesicles which could be observed as a part of agranular endoplasmic reticulum in the cytoplasm.Mitochondria showed the usual size and structure but occurred in larger numbers than mitochondria in A-cells.
Secretory granules in cytoplasm were a rare finding.However, they were frequently found close to cells in the synovial matrix (Fig. 1,2, 3).They were bounded by a smooth membrane and contained either granular or dense homogeneous material.
Intracytoplasmic filaments were regularly observed near the nuclei of B-cells; they were either arranged in parallel to the nuclear envelope or located amongst cytoplasmic organelles '(Fig.2).

Synovial matrix
This term is used to describe an intercellular substance of mesenchymal origin which provides a medium for embedment of synoviocytes.The matrix, as the other tissues of mesenchymal origin, consists of the amorphous and filamentous ground substances.
The structure of synovial matrix. in the horse was different in different layers of the synovial membrane (Figs 1,2, 4, 5).The surface consisted of a 2-3 Jlm layer composed of clusters of aperiodic, irregularly arranged fibrils and condensed amorphous material.This layer included cytoplasmic organelles and secretory granules in the forms of transport vacuoles or dense bodies (Figs 1,2,4,5).The aperiodic fibrils were observed only in the synovial membrane of equine knee joint.They presented as thick bundles either arranged in parallel to the membrane surface (Fig. 4) or located among synoviocytes (Fig. 2).
The synovial matrix enclosing cells (Figs 1,2,5) formed a layer about 4-5 Jlm thick.It was composed of typical collagen fibrils (periodicity, 64 nm) arranged into bundles surrounding single cells; sectioning revealed the bundles cut both longitudinally and transversally.The collagen fibrils were attached to the cell membrane in both A-and Bsynoviocytes (Figs I,2,5).In addition to collagen fibrils, some areas contained an accumulation of aperiodic fibril filling a lot of space.Some differences were found in the arrangement of synovial matrix in regions where synoviocytes were located on the surface (Fig. 3).In the surface layer, the matrix was absent; it was observed in the deeper parts close to subsynovial tissue where it consisted of collagen fibrils arranged in various directions and secretory granules located at cell membranes.The basement membrane was not usually present where the surface layer consisted only of cells (Fig. 3).

Discussion
The synovial membrane, its structure and function has so far received considerable attention (reviewed by Ghadially 1983 ;Horky 1990Horky , 1991) ) and the studies in various mammalian species have been concerned with joint under physiological conditions as well as with those affected by disease (Gangel 1984;Hilbert et al. 1984;Worral et al. 1991).This abundance of information, however, has contained only few data related to the structure of synovial membrane in horses (Pers s on 1971; J oh ans 0 n and Rejn6 1976; Horky 1993).
In describing the cells (synoviocytes) we followed the original classification of Bar I and et al. 1962).A characteristic feature of the synovial membrane in adult horses was, similarly to other mammals, a mixed population of differentiated type A and B cells.Their distribution corresponded to that reported for other mammalian species with the .exception of rats (Was silev 1965).The structures of equine A-and B-cells were also in agreement with the fmdings made in the other mammals with one difference only: B-cells did not show in the cytoplasm the presence of secretory granules desribed by 0 k a d a et al. (1981) and Graabaek (1985).The cytoplasm ofB-cells contained only occasional dense bodies, most probably lysosomes.These bodies were also observed in the synovial matrix close to the membrane surface and it is suggested that they are released into the articular cavity where they may, for instance, take part in the digestion of cartilage intercellular matter (Dingle 1984).In contrast to our earlier observations (Horky 1990(Horky , 1991) ) as well as the literature data, the synovial membrane in the horse did not have the transient A-B cell types.A further difference is the finding of intracytoplasmic ftlaments which are not present in the synovial membrane of cattle (Horky 1984) or pigs in early postnatal periods (Horky 1990).In both cell types, the basement membrane, absent in the pig (Horky 1990(Horky , 1991)), was developed.In B-cells, we failed to detect the penetration of collagen fibrils through the cell membrane of B cells.
The most unusual finding was the occurrence of big bundles of aperiodic ftlaments, 0.2-O.4!lm thick and 5 !lID long, located in a depth of 1-2 !lID under the surface of synovial membrane.They were either arranged in parallel to the synovial surface or bent towards the deeper layer and finish their course among synoviocytes.As far as we are aware such ftlament bundles have never been observed in any mammalian synovial membrane and their origin is the subject of further studies.A comparison of the synovial membrane between knee and astragalar joints did not reveal any differences in its ultrastructure except for the presence of aperiodic fibrils in the synovial matrix of the knee joint.