527
J. Anat. (1977) 123, 2, pp. 527-536 With 9 figures Printed in Great Britain
The surface of dog articular cartilage: A scanning electron microscope study F. N. GHADIALLY, J. A. GHADIALLY, A. F. ORYSCHAK AND N. K. YONG
Department of Pathology, University Hospital, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada (Accepted 25 April 1976) The view that the surface of normal articular cartilage is remarkably smooth is supported by naked eye, light microscopic and transmission electron microscopic studies (Hunter, 1743; Davies, Barnett, Cochrane & Palfrey, 1962; Davies, 1969; Ghadially & Roy, 1969). However, since the advent of scanning electron microscopy, reports have been published claiming that the surface of normal articular cartilage is not smooth but beset by a series of undulations and ridges (called fibre bundles by some), which are believed to play an important role in the functioning and lubrication of joints (McCall, 1968; Gardner & Woodward, 1969; Walker et al. 1969; Redler & Zimny, 1970; Gardner, 1972; Mow, Lai & Redler, 1974). In contrast to this are the scanning electron microscopic studies of Clarke (1971 a, b, c, 1973 a, b) on human cartilage, and our (Ghadially, Ghadially, Oryschak & Yong, 1975) studies on rabbit cartilage, which have failed to reveal such undulations and ridges on cartilage left attached to subchondral bone; however, ridges and undulations are readily produced if cartilage is detached from bone or is cut or injured in certain ways. In both rabbit and man the only constant surface feature detectable with the scanning electron microscope, in cartilage attached to bone, is the occurrence of numerous pits or depressions on the surface. These probably reflect the presence of underlying chondrocytes and their lacunae, but whether such pits occur in vivo, or are the result of preparative procedures whereby shrinkage of underlying chondrocytes leads to a collapse and caving in of the surface, still remains a matter of debate. It seems to us that the articular surface of a number of animal species should be examined to see if these pits are of general occurrence; for, if they are not, one would have difficulty in accepting that these pits are normal features designed to trap synovial fluid and assist in joint lubrication. With this in mind, we have embarked on a study of the articular cartilage of various animal species with the scanning electron microscope. In this paper we report on the surface topography of dog articular cartilage, which is markedly different from that of man and rabbit. Further, we shall show that various changes, including ridges and undulations, can readily be produced when the cartilage is detached from bone, or its integrity is violated by making cuts in its substance.
528
F. N. GHADIALLY AND OTHERS MATERIALS AND METHODS
Ten male and female mongrel dogs, 35-45 lb in weight, were killed under Nembutal anaesthesia. The knee joints were opened and the lower ends of the femora were dissected out taking care that the articular surface was not touched or damaged. After removing muscle and joint capsule, the specimen, which comprised femoral condyles with about 3 cm of attached shaft, was rinsed in four changes of normal saline to remove synovial fluid. In four instances the patella was also collected and treated in a similar fashion. At no stage of the procedure were the articular surfaces allowed to become dry. This was ensured by an assistant whose sole task was to drop normal saline on to the articular surface as and when it seemed necessary. Our experimental design provided a total of 40 femoral condyles and 4 patellae for the following experiments. Experiment 1. Normal cartilage and e.ffect of osmotic shock The object here was to fix some specimens in the usual way but subject others to osmotic shock by exposing them to distilled or tap water, and/or by fixing them in fluids of varying molarity for a period of 15 days. This was achieved as follows: (a) Six femoral condyles and 1 patella were fixed in 3 % glutaraldehyde in 0-1 M cacodylate buffer (518 Osm). (b) Four femoral condyles and 1 patella were soaked in distilled water for 5 minutes and then fixed in 3 % glutaraldehyde in 0-1 M cacodylate buffer (518 Osm). (c) Four condyles were rinsed in tap water for 2 minutes and then fixed in 3 % glutaraldehyde in 0-1 M cacodylate buffer (518 Osm). (d) Four condyles and 1 patella were fixed in 3 % glutaraldehyde in 0-2 M cacodylate buffer (711 Osm). (e) Four condyles and 1 patella were fixed in 3 % glutaraldehyde in distilled water (360 Osm). (f) Four condyles were fixed in 1 5 % glutaraldehyde in distilled water (166 Osm). Experiment 2. Cruciate cuts On the articular surface of 4 condyles a cruciate incision was made with a scalpel: a longitudinal incision was made in the sagittal plane and a transverse incision in the coronal plane at right angles to it. The articular surface was then flushed with saline and fixed in 3% glutaraldehyde in 0-1 M cacodylate buffer for 15 days. Experiment 3. Longitudinal and transverse pieces From the articular surface of 10 condyles a roughly rectangular piece of cartilage (one from each condyle) measuring approximately 10 mm by 7 mm and a little over a millimetre in thickness was removed with the aid of a scalpel. Such pieces of cartilage showed a strong tendency to curl, with the articular surface forming the convex face, in contrast to the rabbit where the tendency to curl was in the opposite direction (Ghadially et al. 1975). These pieces were therefore pinned out on a piece of cork to hold them as flat as possible. The term 'longitudinal piece' will be used to designate those pieces of cartilage where the long axis of the piece corresponded to the sagittal plane of the joint and similarly the term 'transverse piece' will indicate those where the long axis of the piece lay in the coronal plane. All pieces of cartilage were fixed in 3 % glutaraldehyde buffered in 0-1 M cacodylate for 15 days.
S.E.M. of dog articular cartilage surface
-n'>:h*S 0 0
b
529
4
Fig. 1. Numerous humps (H) and a pit (arrow) seen on the surface of dog cartilage attached to bone. Note also an annulus around a hump (arrowhead). x 640.
Processing of tissues After fixation in the various ways described above the specimens were dehydrated in increasing concentrations of alcohol over the course of a week. The major portion of each femoral condyle was then dissected off with a fine fret-saw, rinsed in alcohol and mounted on specimen stubs with 'Electrodag'. The transverse and longitudinal pieces were mounted in a similar fashion. Specimens were vacuum coated with gold in a sputtering device and examined in a scanning electron microscope (Cambridge
Stereoscan). RESULTS
Normal articular surface Unlike the surface of rabbit and human articular cartilage, where pits abound, the surface of dog articular cartilage (covering the femoral condyles and patella) is beset by mounds or elevations which may be described as 'humps' (Fig. 1). While in rare instances a hump seemed to arise from the general articular surface, in most instances the humps appeared to arise from pits, for they had a circular or oval depression around their periphery. In other instances a hump was surrounded by a narrow moat the edge of which was either flush or raised above the articular surface, in the form of an annulus. At times a pair of humps arising from a single pit was seen. Such an appearance supports the idea that these humps and pits reflect the presence of underlying chondrocytes and lacunae. 34
A NA
I23
530
F. N. GHADIALLY AND OTHERS
Fig. 2. A zone of cartilage surface where pits abound, but a few humps arising from the floor of pits are also seen (arrows). x 320.
In addition a few pits without humps in them were seen in virtually every specimen but it must be stressed that practically the entire surface of some specimens and large areas of most specimens were devoid of such pits. However, in a few specimens some zones were found where pits outnumbered the humps (Fig. 2). In one specimen a zone showing bundles of fibres or fine ridges was detected (Fig. 3), while in another the surface appeared to be markedly fibrillated (Fig. 4). These specimens came from the oldest dogs. Osmotic shock experiment Since pits and humps could be attributable to the shrinking or swelling of underlying chondrocytes and lacunae, we studied specimens which had been subjected to a variety of osmotic shocks, like soaking in distilled water, and fixing in hypotonic and hypertonic solutions. However, we could detect no significant differences between these specimens and those collected and processed in the normal way. Cruciate cuts The gaping cruciate cut gave the cartilage a 'hot-cross bun' appearance and divided the cartilage into what may be described loosely as four 'quadrants'. A variety of ridge patterns was seen in the quadrants, the commonest being a system of ridges which ran parallel to one edge of the quadrant and at right angles to another (Fig. 5). Humps and/or pits were seen among the ridges.
S.E.M. of dog articular cartilage surface
531
4 Fig. 3. A zone of cartilage surface from an old dog showing fine ridges or fibre bundles (F). In this region pits and humps are scanty but elsewhere numerous pits and a few humps in pits are seen. x 320. Fig. 4. Fibrillated cartilage surface from an old dog. Characteristic pits and humps are virtually absent. Only one hump (arrow) is evident. x 700. 34-2
532
F. N. GHADIALLY AND OTHERS
6 Fig. 5. A quadrant from a cruciate cut cartilage showing ridges (R) oriented parallel to one edge (A) of the cut and at right angles to the other (B). x 660. Fig. 6. Undulations and ridges found on a longitudinal piece of cartilage. Note humps (H), pits (P) and a series of small nodules suggesting underlying wavy or coiled fibres (arrowheads). x 1280.
*~.'awe_~4*'-
J. Anat. (1977) 123, 2, pp. 527-536 With 9 figures Printed in Great Britain
The surface of dog articular cartilage: A scanning electron microscope study F. N. GHADIALLY, J. A. GHADIALLY, A. F. ORYSCHAK AND N. K. YONG
Department of Pathology, University Hospital, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada (Accepted 25 April 1976) The view that the surface of normal articular cartilage is remarkably smooth is supported by naked eye, light microscopic and transmission electron microscopic studies (Hunter, 1743; Davies, Barnett, Cochrane & Palfrey, 1962; Davies, 1969; Ghadially & Roy, 1969). However, since the advent of scanning electron microscopy, reports have been published claiming that the surface of normal articular cartilage is not smooth but beset by a series of undulations and ridges (called fibre bundles by some), which are believed to play an important role in the functioning and lubrication of joints (McCall, 1968; Gardner & Woodward, 1969; Walker et al. 1969; Redler & Zimny, 1970; Gardner, 1972; Mow, Lai & Redler, 1974). In contrast to this are the scanning electron microscopic studies of Clarke (1971 a, b, c, 1973 a, b) on human cartilage, and our (Ghadially, Ghadially, Oryschak & Yong, 1975) studies on rabbit cartilage, which have failed to reveal such undulations and ridges on cartilage left attached to subchondral bone; however, ridges and undulations are readily produced if cartilage is detached from bone or is cut or injured in certain ways. In both rabbit and man the only constant surface feature detectable with the scanning electron microscope, in cartilage attached to bone, is the occurrence of numerous pits or depressions on the surface. These probably reflect the presence of underlying chondrocytes and their lacunae, but whether such pits occur in vivo, or are the result of preparative procedures whereby shrinkage of underlying chondrocytes leads to a collapse and caving in of the surface, still remains a matter of debate. It seems to us that the articular surface of a number of animal species should be examined to see if these pits are of general occurrence; for, if they are not, one would have difficulty in accepting that these pits are normal features designed to trap synovial fluid and assist in joint lubrication. With this in mind, we have embarked on a study of the articular cartilage of various animal species with the scanning electron microscope. In this paper we report on the surface topography of dog articular cartilage, which is markedly different from that of man and rabbit. Further, we shall show that various changes, including ridges and undulations, can readily be produced when the cartilage is detached from bone, or its integrity is violated by making cuts in its substance.
528
F. N. GHADIALLY AND OTHERS MATERIALS AND METHODS
Ten male and female mongrel dogs, 35-45 lb in weight, were killed under Nembutal anaesthesia. The knee joints were opened and the lower ends of the femora were dissected out taking care that the articular surface was not touched or damaged. After removing muscle and joint capsule, the specimen, which comprised femoral condyles with about 3 cm of attached shaft, was rinsed in four changes of normal saline to remove synovial fluid. In four instances the patella was also collected and treated in a similar fashion. At no stage of the procedure were the articular surfaces allowed to become dry. This was ensured by an assistant whose sole task was to drop normal saline on to the articular surface as and when it seemed necessary. Our experimental design provided a total of 40 femoral condyles and 4 patellae for the following experiments. Experiment 1. Normal cartilage and e.ffect of osmotic shock The object here was to fix some specimens in the usual way but subject others to osmotic shock by exposing them to distilled or tap water, and/or by fixing them in fluids of varying molarity for a period of 15 days. This was achieved as follows: (a) Six femoral condyles and 1 patella were fixed in 3 % glutaraldehyde in 0-1 M cacodylate buffer (518 Osm). (b) Four femoral condyles and 1 patella were soaked in distilled water for 5 minutes and then fixed in 3 % glutaraldehyde in 0-1 M cacodylate buffer (518 Osm). (c) Four condyles were rinsed in tap water for 2 minutes and then fixed in 3 % glutaraldehyde in 0-1 M cacodylate buffer (518 Osm). (d) Four condyles and 1 patella were fixed in 3 % glutaraldehyde in 0-2 M cacodylate buffer (711 Osm). (e) Four condyles and 1 patella were fixed in 3 % glutaraldehyde in distilled water (360 Osm). (f) Four condyles were fixed in 1 5 % glutaraldehyde in distilled water (166 Osm). Experiment 2. Cruciate cuts On the articular surface of 4 condyles a cruciate incision was made with a scalpel: a longitudinal incision was made in the sagittal plane and a transverse incision in the coronal plane at right angles to it. The articular surface was then flushed with saline and fixed in 3% glutaraldehyde in 0-1 M cacodylate buffer for 15 days. Experiment 3. Longitudinal and transverse pieces From the articular surface of 10 condyles a roughly rectangular piece of cartilage (one from each condyle) measuring approximately 10 mm by 7 mm and a little over a millimetre in thickness was removed with the aid of a scalpel. Such pieces of cartilage showed a strong tendency to curl, with the articular surface forming the convex face, in contrast to the rabbit where the tendency to curl was in the opposite direction (Ghadially et al. 1975). These pieces were therefore pinned out on a piece of cork to hold them as flat as possible. The term 'longitudinal piece' will be used to designate those pieces of cartilage where the long axis of the piece corresponded to the sagittal plane of the joint and similarly the term 'transverse piece' will indicate those where the long axis of the piece lay in the coronal plane. All pieces of cartilage were fixed in 3 % glutaraldehyde buffered in 0-1 M cacodylate for 15 days.
S.E.M. of dog articular cartilage surface
-n'>:h*S 0 0
b
529
4
Fig. 1. Numerous humps (H) and a pit (arrow) seen on the surface of dog cartilage attached to bone. Note also an annulus around a hump (arrowhead). x 640.
Processing of tissues After fixation in the various ways described above the specimens were dehydrated in increasing concentrations of alcohol over the course of a week. The major portion of each femoral condyle was then dissected off with a fine fret-saw, rinsed in alcohol and mounted on specimen stubs with 'Electrodag'. The transverse and longitudinal pieces were mounted in a similar fashion. Specimens were vacuum coated with gold in a sputtering device and examined in a scanning electron microscope (Cambridge
Stereoscan). RESULTS
Normal articular surface Unlike the surface of rabbit and human articular cartilage, where pits abound, the surface of dog articular cartilage (covering the femoral condyles and patella) is beset by mounds or elevations which may be described as 'humps' (Fig. 1). While in rare instances a hump seemed to arise from the general articular surface, in most instances the humps appeared to arise from pits, for they had a circular or oval depression around their periphery. In other instances a hump was surrounded by a narrow moat the edge of which was either flush or raised above the articular surface, in the form of an annulus. At times a pair of humps arising from a single pit was seen. Such an appearance supports the idea that these humps and pits reflect the presence of underlying chondrocytes and lacunae. 34
A NA
I23
530
F. N. GHADIALLY AND OTHERS
Fig. 2. A zone of cartilage surface where pits abound, but a few humps arising from the floor of pits are also seen (arrows). x 320.
In addition a few pits without humps in them were seen in virtually every specimen but it must be stressed that practically the entire surface of some specimens and large areas of most specimens were devoid of such pits. However, in a few specimens some zones were found where pits outnumbered the humps (Fig. 2). In one specimen a zone showing bundles of fibres or fine ridges was detected (Fig. 3), while in another the surface appeared to be markedly fibrillated (Fig. 4). These specimens came from the oldest dogs. Osmotic shock experiment Since pits and humps could be attributable to the shrinking or swelling of underlying chondrocytes and lacunae, we studied specimens which had been subjected to a variety of osmotic shocks, like soaking in distilled water, and fixing in hypotonic and hypertonic solutions. However, we could detect no significant differences between these specimens and those collected and processed in the normal way. Cruciate cuts The gaping cruciate cut gave the cartilage a 'hot-cross bun' appearance and divided the cartilage into what may be described loosely as four 'quadrants'. A variety of ridge patterns was seen in the quadrants, the commonest being a system of ridges which ran parallel to one edge of the quadrant and at right angles to another (Fig. 5). Humps and/or pits were seen among the ridges.
S.E.M. of dog articular cartilage surface
531
4 Fig. 3. A zone of cartilage surface from an old dog showing fine ridges or fibre bundles (F). In this region pits and humps are scanty but elsewhere numerous pits and a few humps in pits are seen. x 320. Fig. 4. Fibrillated cartilage surface from an old dog. Characteristic pits and humps are virtually absent. Only one hump (arrow) is evident. x 700. 34-2
532
F. N. GHADIALLY AND OTHERS
6 Fig. 5. A quadrant from a cruciate cut cartilage showing ridges (R) oriented parallel to one edge (A) of the cut and at right angles to the other (B). x 660. Fig. 6. Undulations and ridges found on a longitudinal piece of cartilage. Note humps (H), pits (P) and a series of small nodules suggesting underlying wavy or coiled fibres (arrowheads). x 1280.
*~.'awe_~4*'-
