Articular cartilage: a review and scanning electron microscope study

20 Pages·2005·8.78 MB·English

J. Anat. (1974), 118, 2, pp. 261-280 261
With 18figures
Printedin GreatBritain
Articular cartilage: a review and scanning electron
microscope study
II. The territorial fibrillar architecture
IAN C. CLARKE
Biomechanics Section, Division ofOrthopedics, School ofMedicine,
University ofCalifornia, Los Angeles, U.S.A.
(Accepted 3 June 1974)
INTRODUCTION
Until comparatively recently, the Transmission Electron Microscope (TEM) was
the only tool available for studying the finer morphology of the fibrillar structures
in articular cartilage. With the advent ofthe Scanning Electron Microscope (SEM)
(McCall, 1968) many more research possibilities have been opened up in this field
(Gardner & Woodward, 1969; Inoue, Kodama & Fujita, 1969; Okuda, 1970;
Ohnsorge, Schutt &Holm, 1970;Evans &Clarke, 1971; Puhl, Dustmann & Schulitz,
1971; Highton, 1971; Zimny & Redler, 1972).
However,itisunfortunatethatmuchoftheSEMdatahasnotbeenintegrated with
that obtained previously by TEM and light microscopy. Only by such critical
reviewingcanwebegin to discriminate between artefactsandreal structures (Clarke,
1971a). Themain objective inthispresent study ofthepericellularfibrillarstructures
in articular cartilage was to compare the SEM data with those derived from TEM
studies.
Pericellular morphology in articular cartilage
Concentric zones (Fig. 1) were initially detected around cartilage cells in histo-
logical studies by Morner (1888) and Hansen (1900, 1905) and in describing them a
number ofterms have been used, such as 'chondrinbollar', 'Zellhof', cell territory,
capsule, lacuna and moat (Szirmai, 1969). TEM studies have led to the description
of four regions around each cell as depicted schematically in Fig. 2. There may be
(1) a clear, apparently empty region (c) around the cell into which cytoplasmic
extensions or processes of the cell extend; (2) a moat (m) offine-textured material
usually devoid of collagen fibrils; (3) a territorial region (t) of collagen fibrils
distinct from(4) the fibrils ofthe main interterritorial network (it) byvirtueoftheir
density, orientation and diameter. An example of a pair of cells observed with the
TEM are illustrated in Fig. 3(a).
Table 1 (p.273)summarizespreviouswork.Itisevidentthatstudiesofhumanartic-
ularcartilageareinaminority, andalsothatauthorshaveuseddifferentnamesforthe
zones. In the present study territorial will refer to the distinct pericellular region of
fibrils, interterritorial to the surrounding general network (Zelander, 1959), while
17-2
262
IAN C. CLARKE
Fig. 1. Capsular appearance around cells in articular cartilage. Section stained with Gabbets
methyleneblueandstudiedbytransmittedlight. x100.
Fig. 2. Diagrammaticsketchshowingacartilagecellsurrounded bya 'clear'apparentlyempty
zone(c),amoat(m)ofamorphousorfinefibrillarmaterial,aterritorialmatrix(t)andthemain
interterritorial networks (it).
Territorial architecture ofarticular cartilage 263
,.# zA.,
-
:Il............
44!8 ^
e. .eI'9
1 mi-n 500 nm
3(b) :7~~p
t
500 nrn
3(c)
Fig.3.TEMmicrographsillustratingcellsandsurroundingmatrixwithmoat(m),cellprocesses
(p)andthemainterritorialnetworks(t).(MicrographssuppliedbycourtesyofDrG. Meachim.)
lacuna will be used to describe the cavity in which the cell lies and the immediately
surrounding fibrils.
Cell processes extending into the surrounding matrix have been described in vivo
(Clark & Clark, 1942) but, while detected in most in vitro studies, their apparent
shape and size have varied according to the preparation techniques employed
(Durning, 1958; Smith, Peters & Serafini-Fracassini, 1967; Spycher, Moor &
Ruettner, 1969). Measurements indicate a length of 0-36-09,um and a width of
005-0d1am (Zelander, 1959; Davies et al. 1962; Palfrey & Davies, 1966). The
morphology of the processes are also influenced by site, age and position on the
cell (Davies et al. 1962; Silberberg, 1968; Weiss, Rosenberg & Helfet, 1968) and by
disease (Mitchell & Sheppard, 1970; Ghadially & Kirkaldy-Willis, 1970). Electron-
dense material has frequently been observed on the cell surface and between the
processes. This was described as granular in rabbit and man (Barnett, Cochrane &
Palfrey, 1963; Ghadially, Meachim & Collins, 1965), filamentous in mouse (Silber-
berg, Silberberg & Feir, 1964), and as bundles or tufts in rat (Godman & Porter,
1960).
264
IAN C. CLARKE
In many studies an approximately 0 -1-0,tm wide region oflow electron density
hasbeendescribedseparatingthecell surfacefromthesurroundingmatrix(Zelander,
1959; Godman & Porter, 1960). This region was not apparent in freeze-dried
specimens (Durning, 1958) but was observed in specimens which had been fixed
with osmiumtetroxide(Durning, 1958; Sheldon, 1964; Davies et al. 1962). Henceit
was generally regarded as a fixation artefact (Table 1) created by cell contraction
(Anderson, 1964; Smith et al. 1967) and was thought to contain only embedding
medium (Davies et al. 1962).
The contents ofthe moat which sometimes surrounded either the cell or the clear
region described above has been described as amorphous (Cameron & Robinson,
1958), granular (Davies et al. 1962) and fibrillar (Mitchell & Sheppard, 1970). It
was generally more prominent around cells in the proliferative, hypertrophic and
degenerative zones of epiphyseal cartilage (Cameron & Robinson, 1958; Takuma,
1960), and in the deeper zones ofbothnormal articular cartilage (Davies etal. 1962)
and rheumatoid articular cartilage (Mitchell & Sheppard, 1970). Studies by Barnett
etal. (1963) andAnderson (1964) indicated that these regions were larger and more
common in the degenerate cells ofyoung articular cartilage than in older material,
while Silberberg et al. (1961) suggested that they were associated with ageing of
the tissue.
Although many cartilage cells apparently lie in contact with the main inter-
territorial fibrillar network (Meachim, 1969), many have been described as sur-
rounded by a distinct territorial fibrillar region. In general, when this territorial
region was evident the fibrils in it were unbanded and thinner than elsewhere
(Silberberg et al. 1961; Silberberg, 1968). Silberberg and colleagues (1961) havedes-
cribed threetypes offibril inthecartilage ofmice(Table2, p. 273)whichsuccessively
replace each other as the tissue ages and the fibrils become increasingly closely
packed around thecells. As described by Zelander (1959), thefibrils may be radially
oriented adjacent to the cell, tangentially arranged in the 2-4,tm wide territorial
region and in the form ofa coarse network interterritorially.
MATERIAL AND METHODS
Forty one human femoral heads and 14 acetabula were obtained at post-mortem
from cadavers of subjects aged 32 to 79 years. Specimens were selected from areas
which were either smooth or fibrillated, but which still retained all or part of the
superficial zone offibrils.
The internal fibrous structure ofthe specimens ofarticular cartilage was revealed
by ripping open small blocks of tissue. This was generally done after fixation and
dehydration, but specimens were also fractured at various stages ofpreparation for
comparison. The rupturing force was applied in three alternate directions (Clarke,
1971a) and was reduced by partly undercutting the subchondral bone layer. This
in turn determined the plane offracture.
Blocks of cartilage approximately 12 x 6 mm, with subchondral bone attached,
were sawn from the joints, sketched for identification and orientation, trimmed of
excess bone, and subsequently washed in Ringer's solution to remove synovial fluid
deposits and bone debris. The prepared blocks were fixed in 10% buffered formol
Territorial architecture ofarticular cartilage 265
Fig. 4.Fracturedsurfaceofafemoralspecimeninasubject71 yearsofage,showingtherelative
positions ofthedesignated zonesextendingfromthejoint'ssurface to theunderlyingboneand
marrow spaces. x50.
saline solution for periods of one to four weeks. After fixation, specimens were
washed in water and stored in 700 alcohol ifnot required immediately.
Dehydration was accomplished in ascending strengths of either acetone, or
alcohol, or alcohol followed by amyl acetate or di-ethyl ether, with subsequent
removal of solvents in vacuo. The specimens were generally fractured at this stage,
although freeze-dried specimens were freeze-fractured in Arcton 12 refrigerant prior
to dehydration.
The fractured blocks were usually mounted on stubs with Evostick and coated
with gold palladium, while the freeze-dried material was mounted with Silver Dag
metallic paint and coated with carbon and gold palladium. The specimens were
studied in a Stereoscan 11A scanning electron microscope using gun voltages of
10000, 20000 and 30000.
RESULTS
Fracturedsurfaces
The fractured surfaces extended from the articular surface down to the underlying
trabecular bone (Fig. 4). The striking features in such surfaces were the radial
'ridges' which were orientated perpendicular to the underlying bone but tended to
turn at ornear the articular surface to merge with the superficial zone. It was shown
266 IAN C. CLARKE
6(a) 6b(b) _
Fig. 5. Edges offibrillar layers passing through lacunae intheLSregion (subjects aged 79 and
71 years). x1150.
Fig. 6. Lacunae in UMregion (a) surrounded by concentric layers-in a subject of32 years,
x1300; and (b) withno layers evident-age ofsubject unknown, x2150.
Territorial architecture ofarticular cartilage 267
previously that these 'ridges' were in fact overlapping layers ofapparently random
fibrils (Clarke, 1971a). At low magnifications (x 50) the sites of the cells were
generally visible as black specks because many of the cells were absent, leaving
cavities in the fibrillar network. As previously described (Clarke, 1971a), lacunae
were seldom identifiable in the superficial region (SZ) of the fractured surfaces.
The description of the fractured-surface lacunae is thereforerestricted to theregion
between the lower superficial (LS) and calcified (CZ) zones (Fig. 4). The appearance
of these cellular structures varied considerably throughout each specimen and from
specimen to specimen. In fact each lacuna could virtually have been considered
unique. For this reason the following micrographs (Figs. 5-12) were compiled to
illustrate some ofthe many variations observed.
The majority of the lacunae were situated immediately adjacent to the edges of
the radially running layers of matrix. In fact many layers apparently terminated at
or near the lacunae (Figs. 6a, 7a, 8a), some even coiling around the latter (Fig. 6a),
while other layers seemed to continue between adjacent cavities (Fig. Sa). Less
frequently, lacunae were found lying in the fibrillar network with no sign of radial
layers in their vicinity (Fig. 6b). In the majority ofcases there was no evidence of a
specialized territorial region offibrils. The lacunae appeared simply as cavities in a
network of fibrils which was generally randomly orientated, but in a few instances
paralleled the long-axis ofthe lacunae (Figs. 9a, b). The fibrils in the vicinity ofthe
lacunae varied from 40 to 200 nm thickness, depending on the specimens, but there
was no measurable variation with increasing distance from the cell.
Depending on the manner in which the fracture planes had ruptured through
each group oflacunae, they were observed singly (Figs. 6a, 7b, 8a) or in groups of
two to six or more (Figs. 5a, 8b). The overall size of the lacunae fluctuated in the
5-55 ,um range.
While many ofthe lacunae appeared empty, equally as many contained spherical
bodies with stellate surface contours. These varied from 5 to 20,am in diameter,
sometimes filling the lacunae (Figs. Sa, 8a) but generally only occupying a small
proportion oftheavailablevolume (Figs. 6, 7, 8b). Forcomparisonwith thelacunae
described above in human cartilage, Figs. 10-12 illustrate the corresponding struc-
tures observed in a parallel study of canine cartilage (Hamilton, 1972). In these
specimens, the lacunae contained rounded bodies which took the shape of and
virtually filled the cavities. They had apparently suffered little shrinkage and the
irregular stellate contours described above were not evident. Instead their surfaces
were covered with protrusions approximately 01 ,um thick and 0 1-044,am long
(Figs. lOa, -11, 12b). Several of these bodies had apparently ruptured and it was
possible to examine their internal structures (Fig. lOb). However, there was little
detail to be seen. As in the human specimens there was apparently no connexion
between the contents of the lacunae and the surrounding network, a feature which
may have resulted in the numerous empty lacunae (Figs. Sb, 7a, 8b, 12b).
Figs. 12(a) and (b) show the encroachment of the calcified matrix on the fibrils
and lacunae ofthe deep uncalcified zone.
268
IAN C. CLARKE
_ d~~(~b~)~~~~ ~ ,~
Fig. 7. Lacunae in the MZregion (subjects aged 43 and 68 years). x1450.
Fig. 8. Lacunae in theLMregion (subjects aged 32 and 68 years). x1550.
Territorial architecture ofarticular cartilage 269
* ,-*__~WV, F~ __11 EVA -_ __
Fig. 9. Lacunae in the DZregion (a) adjacenttoasplitinthefractured surface-fibrilsinthis
region appear to be predominantly aligned parallel to the split-68 years, x660, and (b)
adjacent toedge ofafibrillar layer, x1150.
Fig. 10. Rounded bodies evident in lacunae in the DZregion ofyoung adult caninecartilage.
x2500.
270 IAN C. CLARKE
12(a) _ M: ~ 2(b) 1_
Fig. 11. Fine debris (arrowed) lies adjacent to rounded body in DZ lacuna ofyoung adult
canine cartilage. x6400.
Fig. 12(a). Generalview oflacunae at the interface between DZand CZregions (youngadult
canine cartilage). Inserts showlocation ofFigs. 10(a) and 12(b). x600. (b) Lacunae lying at
junction ofDZand CZregions inyoung adultcanine cartilage. x2400.